Protease-deficient Bacillus anthracis

ABSTRACT

The invention relates to a  Bacillus anthracis  ( B. anthracis ) in which more than one secreted protease is inactivated by genetic modification. Such a protease-deficient  B. anthracis  has an improved ability to produce recombinant secreted proteins compared to other bacteria, particularly other  Bacillus . Improvements include production of intact (i.e., mature full-length) proteins, often at high yield. The disclosure provides a  B. anthracis  that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. Also provided is a modified  B. anthracis  comprising such genetic modification transformed with a recombinant molecule encoding a product, as well as methods to prepare and use such  B. anthracis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 and claims the benefit of PCT Application No. PCT/US2012/049321 having an international filing date of Aug. 2, 2012, which designated the United States, which PCT application claimed the benefit of U.S. Provisional Application No. 61/514,384 filed Aug. 2, 2011, and U.S. Provisional Application No. 61/521,617 filed Aug. 9, 2011, the disclosure of each of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronic text file named “6137NIAID-24-PCT_Sequence_Listing_ST25.txt”, having a size in bytes of 195 KB, and created on Aug. 2, 2012. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD

The invention relates to a protease-deficient Bacillus anthracis and its use as a production microorganism to produce stable (i.e., intact) proteins, including recombinant secreted proteins.

BACKGROUND

Species of Bacillus, such as Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, are attractive microorganisms for recombinant protein production in view of their fast growth rate, high yield, and ability to secrete produced products directly into the medium. Bacillus anthracis is also attractive in view of its ability to produce anthrax toxin and ability to fold proteins correctly. However, the attractiveness of Bacillus is reduced in view of the large quantities of extracellular proteases that the microorganisms secrete into the medium, leading to protein degradation, and the fact that they form spores.

The Gram-positive bacterial pathogen Bacillus anthracis (also referred to herein as B. anthracis) secretes high levels of the three proteins that are collectively termed anthrax toxin: protective antigen (PA), edema factor (EF), and lethal factor (LF), when grown under conditions thought to mimic those in an infected animal host. PA is a receptor-binding component which acts to deliver LF and EF to the cytosol of eukaryotic cells; EF is a calmodulin-dependent adenylate cyclase, and LF is a zinc metalloprotease that cleaves most members of the mitogen-activated protein kinase kinase family; see, for example, Leppla S H et al., 2000, in Anthrax toxin, bacterial protein toxins, 445-472, Aktories K et al., (Eds.), Springer, Berlin; Moayeri M et al., 2011, Anthrax toxins, Bacillus anthracis and Anthrax, 121-156, Bergman (Ed.), John Wiley & Sons, Inc., Hoboken, N.J.; Moayeri M et al., 2009, Mol. Aspects. Med. 30, 439-455; Young J A et al., 2007, Annu Rev. Biochem. 76, 243-265. PA, EF, and LF are encoded on virulence plasmid pXO1 by pag, cya, and lef, respectively. Virulence plasmid pXO2 encodes proteins required for capsule formation and depolymerization. B. anthracis that lack one or both virulence plasmids are typically attenuated in most animal hosts. Single PA, EF, and LF components are non-toxic; a combination of one PA and at least two EFs, at least two LFs, or mixtures of EF and LF are required for toxicity.

Because anthrax pathogenesis is highly dependent on the actions of the anthrax toxin proteins, vaccine and therapeutic development efforts have focused on countering toxin action, typically by generating antibodies to PA. The anthrax vaccine currently licensed in the USA, and developed almost 50 years ago (see, e.g., Puziss M et al., 1963, J. Bacteriol. 85, 230-236), consists of a partially purified culture supernatant of a protease-deficient B. anthracis strain (V770-NP1-R). PA is the most abundant protein and the key immunogen in this vaccine. Efforts to produce a recombinant PA vaccine from B. anthracis by scale-up of an established process (see, e.g., Farchaus J W et al., 1998, Appl. Environ. Microbiol. 64, 982-991) appear to have been hampered by instability of the final product.

While the toxin components can be purified as recombinant proteins from B. anthracis culture supernatants (see, e.g., Farchaus J W et al., ibid.; Varughese M et al., 1999, Infect. Immun. 67, 1860-1865; Singh Y et al., 1991, J. Biol. Chem. 266, 15493-15497; Park S et al., 2000, Protein Expr. Purif. 18, 293-302), the integrity and yields are limited by the B. anthracis proteolytic enzymes that are co-secreted.

Two extracellular proteases are reported to be abundant in the B. anthracis secretome: NprB (GBAA_0599), neutral protease B, a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species; and InhA1 (GBAA_1295), immune inhibitor A1, a homolog of the immune inhibitors A from other members of the Bacillus cereus group (see, e.g., Antelmann H et al., 2005, Proteomics 5, 3684-3695; Chitlaru T et al., 2006, J. Bacteriol 188, 3551-3571; Chung M C et al., 2006, J. Biol. Chem. 281, 31408-31418. These two proteases contain zinc-binding motifs typical for the zincin tribe of metallopeptidases (His-Glu-Xxx-Xxx-His (SEQ ID NO:31)) and belong, respectively, to the M4 and M6 families of metalloproteases according to the MEROPS database, Wellcome Trust Sanger Institute (see e.g., website of Wellcome Trust Sanger Institute).

A third metalloprotease, camelysin (GBAA_1290), belonging to the M73 family is found in the secretome of several B. anthracis strains. This protease is similar to the camelysin of B. cereus, a novel surface metalloprotease; see, e.g., Grass G et al, 2004, Infect. Immun. 219-228.

B. anthracis also contains a gene encoding InhA2 metalloprotease (GBAA_0672, M6 family), although it is not known whether this protease is expressed and secreted. This gene is an ortholog of the InhA1 described above (68% amino acid identity). Similarly, the genome of B. anthracis also contains genes encoding TasA (GBAA_1288, M73 superfamily), which is an ortholog of camelysin (60% amino acid identity), and MmpZ (GBAA_3159, ZnMc superfamily), which is a putative extracellular zinc-dependent matrix metalloprotease, a member of the metzincin clan of metallopeptidases. This clan is characterized by an extended zinc-binding motif (His-Glu-Xxx-Xxx-His-Xxx-Xxx-Gly/Asn-Xxx-Xxx-His/Asp (SEQ ID NO:32)) (see, e.g., Gomis-Ruth, F X, 2009, J. Biol. Chem. 284, 15353-15357).

Bacillus subtilis strains having more than one protease inactivated have been produced and analyzed. For example, Wu X C et al., 1991, J. Bacteriol. 173, 4952-4958 produced a B. subtilis strain deficient in six extracellular proteases (WB600), namely neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B. WB600 showed only 0.32% of the extracellular protease activity of wild-type B. subtilis strains. Kurashima K et al., 2002, J. Bacteriol. 184, 76-81, expressed apparently intact Clostridium cellulovorans EngB cellulase in a B. subtilis strain deficient in eight proteases (WB800). WB800 was derived from WB600 through inactivation of VpR protease and cell wall protease WprA. WB700 was derived from WB600 through inactivation of VpR (see, e.g., Wu et al, 2002, Appl. Environ. Microbiol. 68, 3261-3269).

There have been reports of inactivation of certain individual B. anthracis proteases: Inactivation of B. anthracis NprB led to reduced proteolysis of casein (see, e.g., Pomerantsev A P et al., 2006, Infect. Immun. 74, 682-693. Inactivation of InhA1 indicated that coagulation of human blood by B. anthracis required InhA1 for proteolytic activation of prothrombin and factor X (see, e.g., Kastrup C J et al., 2008, Nat. Chem. Biol. 4, 742-750. However, production of anthrax toxin proteins in both of these strains led to protein degradation over time, albeit at a later time than production in B. anthracis A35.

There remains a need for a B. anthracis that can produce large amounts of stable (i.e., intact) proteins, such as anthrax toxin proteins PA, EF, and LF.

SUMMARY

The invention relates to a B. anthracis in which more than one secreted protease is inactivated by genetic modification. Such a protease-deficient B. anthracis has an improved ability to produce recombinant secreted proteins compared to other bacteria, particularly other Bacillus. Improvements include production of intact (i.e., mature full-length) proteins, often at high yield.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases. An example of a M4 protease is NprB. Examples of M6 proteases include InhA1 and InhA2. Examples of M73 proteases are camelysin and TasA. An example of a ZnMc protease is MmpZ.

One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease and a genetic modification that inactivates InhA1 protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, and a genetic modification that inactivates MmpZ protease.

The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof.

The disclosure also provides that any of these protease-deficient B. anthracis can lack SinR and SinI regulatory proteins. The disclosure also provides that any of these protease-deficient B. anthracis can be sporulation deficient. The disclosure also provides that any of these protease-deficient B. anthracis can lack one or more virulence plasmids. Genetic modification of any of these protease-deficient B. anthracis can be selected from the group consisting of deletion, insertion, inversion, substitution, derivatization, and combinations thereof, wherein such genetic modification affects one or more nucleotides in a gene encoding the protein. In one embodiment, genetic modification comprises deletion of one or more nucleotides in a gene encoding a protein, insertion of one or more nucleotides in a gene encoding a protein, or a combination thereof.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; a genetic modification that inactivates InhA1 protease, wherein the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; and a genetic modification that inactivates Spo0A protein, wherein the inactivated Spo0A protein is encoded by a genetically modified spo0A gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXO1 and pXO2. One embodiment is a B. anthracis having the identifying characteristics of BH460, deposited under ATCC Accession No. PTA-12024. One embodiment is B. anthracis BH460, deposited under ATCC Accession No. PTA-12024.

The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification that inactivates a protease selected from the group consisting of a protease of the transglutaminase-like superfamily, a protease of peptidase family S8, and a combination thereof. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One example of a transglutaminase-like superfamily protease is CysP1 protease. One example of a peptidase family S8 protease is VpR protease. In one embodiment, the protease of the transglutaminase-like superfamily is CysP1 protease. In one embodiment, the protease of peptidase family S8 is VpR protease. In one embodiment, the protease of the transglutaminase-like superfamily is CysP1 protease, and the protease of peptidase family S8 is VpR protease. As used herein, CysP1 protease is also referred to as BA1995 protease.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysP1 protease, and a genetic modification that inactivates VpR protease. In one embodiment, the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; the inactivated CysP1 protease is encoded by a genetically modified cysP1 gene at locus GBAA_1995 of the B. anthracis; and the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA_4584 of the B. anthracis.

The disclosure provides that any of the protease-deficient B. anthracis can also comprise a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. As used herein, SprA protease is also referred to as BA5414 protease.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysP1 protease, a genetic modification that inactivates VpR protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

The disclosure also provides that any of these protease-deficient B. anthracis can lack SinR and SinI regulatory proteins. The disclosure also provides that any of these protease-deficient B. anthracis can be sporulation deficient. The disclosure also provides that any of these protease-deficient B. anthracis can lack one or more virulence plasmids. Genetic modification of any of these protease-deficient B. anthracis can be selected from the group consisting of deletion, insertion, inversion, substitution, derivatization, and combinations thereof, wherein such genetic modification affects one or more nucleotides in a gene encoding the protein.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; a genetic modification that inactivates InhA1 protease, wherein the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; a genetic modification that inactivates CysP1 protease, wherein the inactivated CysP1 protease is encoded by a genetically modified cysP1 gene at locus GBAA_1995 of the B. anthracis; a genetic modification that inactivates VpR protease, wherein the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA_4584 of the B. anthracis; and a genetic modification that inactivates Spo0A protein, wherein the inactivated Spo0A protein is encoded by a genetically modified spo0A gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXO1 and pXO2. One embodiment is a B. anthracis having the identifying characteristics of BH480, deposited under ATCC Accession No. PTA-13162. One embodiment is B. anthracis BH480, deposited under ATCC Accession No. PTA-13162.

The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases and to inactivate a protease of the M6 family of metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, and to inactivate a protease of the M73 family of metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, and to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases, to inactivate a protease of the transglutaminase-like superfamily, and to inactivate a protease of peptidase family S8.

The disclosure provides a method to produce any B. anthracis of the embodiments. Such a method comprises culturing the B. anthracis in a medium; and recovering the B. anthracis.

The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment of a product is a product selected from the group consisting of a protein, an amino acid, a nucleic acid molecule, a compound produced via a recombinant biosynthetic pathway, a small molecule, a drug, a vitamin, a drug conjugate, and a peptide nucleic acid conjugate. In one embodiment, such a product is a toxin. Examples of toxins include an anthrax toxin, a cholera toxin, a diphtheria toxin, a hemolysin, and a ricin. Additional examples of toxins include a Pseudomonas toxin, a Haemophilus ducreyi toxin, an Escherichia coli toxin, and a ribosome-inactivating protein (RIP) toxin.

The disclosure provides a recombinant molecule that comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. Examples of recombinant molecules include a recombinant molecule comprising a nucleic acid sequence selected from the group consisting of a recombinant molecule comprising nucleic acid sequence SEQ ID NO:62, a recombinant molecule comprising nucleic acid sequence SEQ ID NO:65, a recombinant molecule comprising nucleic acid sequence SEQ ID NO:67, and a recombinant molecule comprising nucleic acid sequence SEQ ID NO:69.

One embodiment is a recombinant molecule comprising a pSJ115 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:91. One embodiment is a recombinant molecule comprising a pYS5 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:92. One embodiment is a recombinant molecule selected from the group consisting of recombinant molecule pSJ136EF-His, recombinant molecule pSJ136EF-Cys, and recombinant molecule pSJ136EF-NEHY. The disclosure also provides a protein comprising an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO:93, amino acid sequence SEQ ID NO:94, and amino acid sequence SEQ ID NO:95.

The disclosure provides a method to produce a product comprising: culturing a modified B. anthracis of the embodiments in a medium to produce the product; and recovering the product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides sequences of truncated and inactivated B. anthracis proteases. FIG. 1A provides sequences of truncated and inactivated B. anthracis NprB and InhA1 proteases. FIG. 1B provides sequences of truncated and inactivated B. anthracis InhA2, TasA, camelysin, and MmpZ proteases, as well as sequences used to produce the deletion of a B. anthracis genome spanning a portion of tasA through a portion of inhA1, as described in the Examples. Protease genes were inactivated using a procedure that results in the insertion of the 34-bp pair loxP sequence (underlined in the nucleotide sequences) flanked by several endonuclease restriction sites (positions not indicated). The large region between the TasA and InhA1 genes was replaced by a loxP sequence using the same procedure. For each protease, the amino acid and nucleotide sequences shown in bold in the upper section identify the final three amino acids retained from the original protease. The amino acid sequences following this trimer are nonsense and/or out of frame translations encoded by the restriction sites and loxP sequences; translation terminates at the codons indicated by asterisks. The lower section of each of InhA2, TasA, camelysin, and MmpZ protease shows the entire amino acid sequence (including signal sequence) for each respective protease. The frame shift that occurs following the three amino acids shown in each upper section causes only the underlined portion of the respective original amino acid sequence to be translated. The zinc-binding active site sequences for NprB and InhA1 are shown in bold in FIG. 1A. Identical zinc-binding active site sequences are shown in bold in the InhA2 and MmpZ amino acid sequences in FIG. 1B. For the large TasA-InhA1 deletion, truncation of the TasA protein occurs at the same site as for the single TasA deletion. The deleted DNA sequence extends through the corresponding sequence encoding the first 402 amino acids of InhA1 (not indicated). The remaining DNA begins with the sequence that would encode the last 393 amino acids of InhA1, which begins with the sequence IMSGGSWAGKIAGTTPTSFS (SEQ ID NO:33) (dashed underlining). FIG. 1C provides sequences of truncated and inactivated B. anthracis CysP1 and VpR proteases. These protease genes were inactivated using a procedure that results in replacement of at least a portion of the genes with a 48-bp FRT-site (underlined in the nucleotide sequences) using Flp recombinase.

FIG. 2 provides growth curves of certain B. anthracis strains and production analyses of edema factor (EF), anthrolysin O (ALO), protective antigen (PA), lethal factor (LF), and camelysin by these strains. Growth curves in LB medium are shown for ten B. anthracis strains over 24 h. Western blot analyses of EF, ALO, PA, LF, and camelysin at various time points are shown for each strain. The most slowly migrating band in each set of blots appears to correspond to the respective intact, or nearly intact, protein.

FIG. 3 provides production analyses of LF and ALO by certain genetically modified strains, some of which had a deficient protease function complemented by a plasmid encoding active protease. Western blot analyses are shown of (A) LF production by A35ΔMmpZ compared to A35ΔMmpZC and (B) ALO production by A35TM compared to A35TMC. (C at the end of the strain name indicates a complemented strain.) Numbers on the top of each lane indicate time in hours at which samples were taken.

FIG. 4 provides a comparison of EF proteins purified from E. coli BL21(DE3), B. anthracis BH450 (with a genetic modification in nprB and in spo0A), and B. anthracis BH460. A) SDS-PAGE analysis of the EF proteins. M—molecular mass markers (Page Ruler Unstained Protein Ladder, Fermentas). B) Electron Spray Ionization—Mass Spectra (ESI-MS) of the EF samples shown in A). The Y-axis represents relative abundance and the X-axis represents mass/charge ratio (m/z). C) Comparison of the experimental molecular masses of the EF samples extrapolated from ESI-MS data to the molecular masses calculated from the amino acid sequences. Relative abundances of the resulting components from the ESI-MS data are shown in parentheses. The final line shows differences between experimental and calculated masses.

FIG. 5 provides EF activity analyses. A) cAMP production by different EF preparations was measured following treatment of RAW264.7 cells for 1 h with a range of EF concentrations and a set PA concentration (250 ng/ml). B) Potency of EF prepared from BH460 was compared to EF prepared from BH450 or from E. coli BL21(DE3) in Balb/cJ mice challenged with either 25 μg EF+25 μg PA (for EF made from BH450, BH460, or E. coli, respectively) or 50 μg EF+50 μg PA (for EF made from BH450 only).

FIG. 6 provides a schematic map of recombinant molecule pSJ136EFOS (SEQ ID NO:62), which comprises a nucleic acid molecule encoding mature EF with a PA signal sequence (SEQ ID NO:63) operatively linked to a pag promoter. The pag promoter spans approximately nucleotides 3496-3658 of SEQ ID NO:62. The PA signal sequence is encoded by nucleotides beginning at nucleotide 3659 of SEQ ID NO:62. The EF mature protein (SEQ ID NO:64) is encoded by nucleotides beginning at nucleotide 3746 of SEQ ID NO:62. The E. coli ORI (not shown) spans approximately nucleotides 6286-6755 of SEQ ID NO:62.

FIG. 7 provides a schematic map of recombinant molecule pSW4-HBL L1 His (SEQ ID NO:65), which comprises a nucleic acid molecule encoding HBL L1 His operatively linked to a pag promoter. The HBL L1 His protein (SEQ ID NO:66) is encoded by nucleotides beginning at nucleotide 6611 of SEQ ID NO:65.

FIG. 8 provides a schematic map of recombinant molecule pSW4-HBL L2 His (SEQ ID NO:67), which comprises a nucleic acid molecule encoding HBL L2 His operatively linked to a pag promoter. The HBL L2 His protein (SEQ ID NO:68) is encoded by nucleotides beginning at nucleotide 3660 of SEQ ID NO:67.

FIG. 9 provides a schematic map of recombinant molecule pSW4-HBL B His (SEQ ID NO:69), which comprises a nucleic acid molecule encoding HBL B His operatively linked to a pag promoter. The HBL B His protein (SEQ ID NO:70) is encoded by nucleotides beginning at nucleotide 2 of SEQ ID NO:69.

FIG. 10 provides native Phast gel (native 8-25% acrylamide gradient) analysis of production of EF proteins EFOS (lane 4), EF-His (lane 6), EF-Cys (lane 7), and EF-NEHY (lane 8) by B. anthracis BH480 transformed with recombinant molecule pSJ136EFOS, B. anthracis BH480 transformed with recombinant molecule pSJ136EF-His, B. anthracis BH480 transformed with recombinant molecule pSJ136EF-Cys, or B. anthracis BH480 transformed with recombinant molecule pSJ136EF-NEHY, respectively. Lanes 1 and 2 show production of PA-U2f and PA-U7f by modified B. anthracis BH480. Lane 3 shows production of mature lethal factor (LF-OS) by B. anthracis BH480 transformed with pSJ115-LF-OS. Lane 5 shows production of Hfq1-FLAG by B. anthracis BH480 transformed with pSJ136 Hfq1-FLAG.

FIG. 11 provides SDS Phast gel analysis of production of LFnBlaY (SEQ ID NO:91) and PA-SNKE-deltaFF-E308D (SEQ ID NO:92) proteins by B. anthracis BH480 transformed with recombinant molecules encoding either LFnBlaY or PA-SNKE-deltaFF-E308D, respectively. Lane 1: SDS molecular weight marker mix. Lane 2: PA-SNKE-deltaFF-E308D produced by B. anthracis BH480 transformed with a pYS5 plasmid encoding PA-SNKE-deltaFF-E308D; Lane 3: LF-BLA, recombinantly produced in E. coli; Lanes 4 and 5: LFnBlaY produced by B. anthracis BH480 transformed with a pSJ115 plasmid encoding LFnBlaY.

DETAILED DESCRIPTION

The present disclosure describes the novel finding that genetic modifications of Bacillus anthracis (B. anthracis) that inactivate more than one secreted protease provide a protease-deficient B. anthracis with an improved ability to produce recombinant secreted proteins compared to other bacteria, e.g., other B. anthracis strains, including those with a single inactivated secreted protease. Improvements include production of intact (i.e., mature full-length) proteins, often at high yield. For example, one embodiment is B. anthracis BH460. BH460 has genetic modifications that inactivate six proteases, is sporulation-deficient, and is free of the virulence plasmids. This strain provides an improved host for production of recombinant proteins. As an example, EF produced from BH460 is highly active, whereas previous B. anthracis host strains produced truncated EF proteins having low potency. The ability to produce an intact EF allows EF to be a component of a recombinant anthrax vaccine. As another example, one embodiment is B. anthracis BH480. BH480 has genetic modifications that inactivate eight proteases, is sporulation-deficient, and is free of the virulence plasmids. This strain also provides an improved host for production of recombinant proteins as shown herein.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an” entity refers to one or more of that entity. For example, a genetic modification refers to one or more genetic modifications. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly the terms “comprising”, “including” and “having” can be used interchangeably.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The disclosure provides a B. anthracis comprising genetic modifications that inactivate more than one protease. In one embodiment, such inactivated proteases are inactivated secreted proteases. As used herein the phrase “a B. anthracis comprising a genetic modification that inactivates a protease” refers to a B. anthracis strain (also referred to as a B. anthracis organism or a B. anthracis microorganism) in which at least one of its proteases has been inactivated by at least one genetic modification. It is to be noted that the terms inactivated, inactive, defective, and deficient can be used interchangeably.

As used herein, a protease (also referred to as a proteinase or a peptidase) is any enzyme that conducts proteolysis, i.e., an enzyme that initiates protein catabolism by hydrolyzing the peptide bonds that link amino acids together in a chain to form a protein. A protein is any compound that has two or more amino acids linked together by a peptide bond between the carboxyl and amino groups of adjacent amino acids; as such, the term protein includes polypeptides and peptides. A secreted protease is a protease that is secreted from the cell (e.g., B. anthracis) that produces it. An inactivated protease is a protease that no longer functions to hydrolyze peptide bonds. An inactivated protease can have amino acids that have been deleted (e.g., to form a truncated protein), inserted, inverted, substituted, derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), or subjected to any other change known to those skilled in the art in order to deactivate the protease. An inactivated protease can be produced by effecting deletion, insertion, inversion, substitution, derivatization, and/or or any other change of amino acids at the amino acid level in order to deactivate the protease. Alternatively, inactivation can occur at the nucleic acid level by genetic modification (also referred to herein as mutation). Genetic modification includes deletion, insertion, inversion, substitution, derivatization, and/or any other change known to those skilled in the art of one or more nucleotides in a gene encoding the protease such that the genetically modified nucleic acid does not encode an active protease. In some embodiments, no protease is produced at all. In some embodiments, the encoded protease is truncated. In one embodiment, genetic modification comprises deletion of one or more nucleotides in a gene encoding a protease. In one embodiment, such a deletion comprises Cre-loxP gene knockout, a technique further described in the Examples herein. One embodiment is a conditionally inactivated protease, which can be produced, for example, using inducible antisense or anti-parallel loxP sites bracketing the structural gene encoding such a protease so that the Cre recombinase flips the gene between active and inactive forms. In one embodiment, a protease gene is inactivated via a Saccharomyces cerevisiae Flp-FRT recombinase system.

One embodiment is a B. anthracis comprising genetic modification that inactivates at least two secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least three B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least four B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least five B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least six B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least seven B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least eight B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least nine B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least ten B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least eleven B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least twelve B. anthracis secreted proteases

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. The M4 and M6 families of metalloproteases are as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and an inactivated protease of the M6 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and at least two inactivated proteases of the M6 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and two inactivated proteases of the M6 family.

A non-limiting example of a protease of the M4 family of metalloproteases is B. anthracis NprB protease, also referred to herein as NprB. In one embodiment a genetic modification that inactivates a protease of the M4 family of metalloproteases is a genetic modification that inactivates NprB. Non-limiting examples of a protease of the M6 family of metalloproteases are B. anthracis InhA1 protease and B. anthracis InhA2 protease (also referred to herein as InhA1 and InhA2, respectively). In one embodiment a genetic modification that inactivates a protease of the M6 family of metalloproteases is a genetic modification that inactivates a protease of the M6 family selected from the group consisting of InhA1, InhA2, and combinations thereof. As such, genetic modification can inactivate InhA1, genetic modification can inactivate InhA2, or genetic modification can inactivate both InhA1 and InhA2. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB and InhA1. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB and InhA2. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, and InhA2.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases. The M73 family of metalloproteases is as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. Surprisingly, a B. anthracis comprising such genetic modifications produced significantly more intact secreted protein than did strains having inactivated proteases of the M4 and/or M6 families of metalloproteases but an active protease of the M73 family. Such improved production is exemplified in the Examples.

One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and at least two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, and at least two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, and two inactivated proteases of the M73 family.

Non-limiting examples of a protease of the M73 family of metalloproteases are B. anthracis camelysin protease and B. anthracis TasA protease (also referred to herein as camelysin and TasA, respectively.) In one embodiment a genetic modification that inactivates a protease of the M73 family of metalloproteases is a genetic modification that inactivates a protease of the M73 family selected from the group consisting of camelysin, TasA, and combinations thereof. As such, genetic modification can inactivate camelysin, genetic modification can inactivate TasA, or genetic modification can inactivate both camelysin and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, camelysin, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, camelysin, and TasA.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The ZnMc superfamily of zinc-dependent metalloproteases is as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, at least two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, at least two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily.

A non-limiting example of a protease of the ZnMc superfamily of metalloproteases is B. anthracis MmpZ protease, also referred to herein as MmpZ. In one embodiment a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases is a genetic modification that inactivates MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, camelysin, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA1, InhA2, camelysin, TasA, and MmpZ.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates at least two proteases, wherein the proteases are selected from the group consisting of a protease of the M4 family of metalloproteases, a protease of the M6 family of metalloproteases, a protease of the M73 family of proteases, and a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure encompasses any combination of two or more such inactivated proteases. One or more genetic modifications can lead to the inactivation of such two or more proteases. For example, a deletion spanning at least a portion of two genes that encode such proteases can effect at least two inactivated proteases. One embodiment is a B. anthracis comprising a genetic modification that inactivates at least two proteases, wherein the proteases are selected from the group consisting of NprB, InhA2, TasA, camelysin, InhA1, and MmpZ.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, and a genetic modification that inactivates MmpZ protease. It is to be appreciated that a genetic modification that inactivates one protease can be the same genetic modification that inactivates one or more additional proteases. For example, a genetic modification that deletes a region of the B. anthracis genome that spans from at least a portion of the tasA gene through at least a portion of the inhA1 gene is an example of a genetic modification that inactivates a TasA protease, a genetic modification that inactivates a camelysin protease, and a genetic modification that inactivates an InhA1 protease. In one embodiment, such a deletion spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17. SEQ ID NO:9 and SEQ ID NO:17 encode wild-type TasA and InhA1, respectively. Such a deletion also deletes the genes encoding regulatory proteins SinR and SinI.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease and a genetic modification that inactivates InhA1 protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease and InhA1 proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; and (b) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, camelysin, and InhA1 proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; (b) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; and (c) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, TasA, camelysin, and InhA1 proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; (b) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; (c) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; and (d) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhA1 protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, InhA2, TasA, camelysin, and InhA1 proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; and (e) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, and a genetic modification that inactivates MmpZ protease.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1 and MmpZ proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; (e) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; and (f) the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1 and MmpZ proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the inactivated InhA2 protease is encoded by an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the inactivated TasA protease is encoded by a tasA gene that encodes an inactivated TasA comprising amino acid sequence SEQ ID NO:10; (d) the inactivated camelysin protease is encoded by a calY gene that encodes an inactivated camelysin comprising amino acid sequence SEQ ID NO:14; (e) the inactivated InhA1 protease is encoded by an inhA1 gene that encodes an inactivated InhA1 comprising amino acid sequence SEQ ID NO:18; and (f) the inactivated MmpZ protease is encoded by a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22. In one embodiment, the amino acid sequence of the encoded inactivated NprB protease is SEQ ID NO:2. In one embodiment, the amino acid sequence of the encoded inactivated InhA2 protease is SEQ ID NO:6. In one embodiment, the amino acid sequence of the encoded inactivated TasA protease is SEQ ID NO:10. In one embodiment, the amino acid sequence of the encoded inactivated camelysin protease is SEQ ID NO:14. In one embodiment, the amino acid sequence of the encoded inactivated InhA1 protease is SEQ ID NO:18. In one embodiment, the amino acid sequence of the encoded inactivated MmpZ protease is SEQ ID NO:22.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1 and MmpZ proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhA1 protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17 (such a deletion also deletes genes encoding SinR and SinI); and (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, and MmpZ proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB consisting of amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 consisting of amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhA1 protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17 (such a deletion also deletes genes encoding SinR and SinI); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ consisting of amino acid sequence SEQ ID NO:22.

In some embodiments, B. anthracis of the disclosure can also include genetic modifications that inactivate regulatory protein SinR, regulatory protein SinI, or both SinR and SinI. SinR and SinI are encoded by genes located at GBAA_1292 and GBAA_1293, respectively, of the B. anthracis genome.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases and a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof. BA1995, VpR and BA5414 proteases have been identified as secreted proteases present in the supernatant of B. anthracis in which NprB, InhA2, TasA, camelysin, InhA1, and MmpZ proteases have been inactivated. BA1995 is encoded by a gene located at locus GBAA_1995 of B. anthracis (see, e.g., Sastalla I et al., 2010, Microbiology 156, 2982-2993). VpR, or BA4584, is a minor extracellular protease that is encoded by the vpR gene located at locus GBAA_4584 of B. anthracis. BA 5414, is a serine protease that is encoded by a gene located at locus GBAA_5414 of B. anthracis.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof.

Additional secreted proteases, the inactivation of which would further improve protein production by B. anthracis of the embodiments, can be identified using techniques known to those skilled in the art. For example, a protease-deficient B. anthracis of the embodiments can be cultured in a medium, and the resulting medium, or supernatant produced from the medium, can be tested for protease activity against desired proteins being produced by such B. anthracis. Protease(s) responsible for such activity can be isolated, sequenced, and the sequence(s) compared to the genomic map of B. anthracis in order to identify the gene(s) that encode it (them). Such gene(s) can then be inactivated using techniques described herein.

The disclosure provides that any of the B. anthracis of the embodiments can be sporulation-deficient. That is, any of the B. anthracis described herein can also comprise a genetic modification that prevents sporulation, such as a genetic modification that inactivates the Spo0A protein. The Spo0A protein, a key regulator of sporulation in Bacillus (see, e.g., Molle V et al., 2003, Molec. Microbiol. 50, 1683-1701), is encoded by the spo0A gene, located at locus GBAA_4394 of B. anthracis. B. anthracis that are unable to sporulate are advantageous as production organisms.

The disclosure also provides that any of the B. anthracis of the embodiments can be deficient in one or more virulence plasmids. One embodiment is a B. anthracis of the embodiments that is pXβ1−, i.e., the B. anthracis lacks virulence plasmid pXO1. One embodiment is a B. anthracis of the embodiments that is pXO1−, pXO2−; i.e., the B. anthracis lacks virulence plasmids pXO1 and pXO2. One embodiment is a B. anthracis of the embodiments that is pXO1−, pXO2+.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; a genetic modification that inactivates InhA1 protease, wherein the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; and a genetic modification that inactivates Spo0A protein, wherein the inactivated Spo0A protein is encoded by a genetically modified spo0A gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXO1 and pXO2.

The disclosure provides a Bacillus anthracis having the identifying characteristics of BH460, deposited under ATCC Accession No. PTA-12024. A deposit of Bacillus anthracis BH460 has been made on Aug. 9, 2011 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, under ATCC accession number PTA-12024. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. Identifying characteristics of BH460 include (a) inactivated NprB, InhA2, TasA, camelysin, InhA1, and MmpZ proteases, (b) sporulation-deficiency, and (c) lack of virulence plasmids pXO1 and pXO2. One embodiment of the disclosure is Bacillus anthracis BH460.

The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification that inactivates a protease selected from the group consisting of a protease of the transglutaminase-like superfamily, a protease of peptidase family S8, and a combination thereof. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, and a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, and an inactivated protease of the transglutaminase-like superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, and an inactivated protease of peptidase family S8. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, an inactivated protease of the transglutaminase-like superfamily, and an inactivated protease of peptidase family S8.

A non-limiting example of a protease of the transglutaminase-like superfamily is B. anthracis CysP1 protease, also referred to herein as CysP1. In one embodiment, a genetic modification that inactivates a protease of the transglutaminase-like superfamily is a genetic modification that inactivates CysP1. A non-limiting example of a protease of peptidase family S8 is B. anthracis VpR protease (also referred to herein as VpR). In one embodiment, a genetic modification that inactivates a protease of peptidase family S8 is a genetic modification that inactivates VpR. As such, genetic modification can inactivate CysP1, genetic modification can inactivate VpR, or genetic modification can inactivate both CysP1 and VpR. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, and CysP1. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, and VpR. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysP1 protease, and a genetic modification that inactivates VpR protease. It is to be appreciated that a genetic modification that inactivates one protease can be the same genetic modification that inactivates one or more additional proteases. For example, as disclosed above, a genetic modification that deletes a region of the B. anthracis genome that spans from at least a portion of the tasA gene through at least a portion of the inhA1 gene is an example of a genetic modification that inactivates a TasA protease, a genetic modification that inactivates a camelysin protease, and a genetic modification that inactivates an InhA1 protease. In one embodiment, such a deletion spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17. SEQ ID NO:9 and SEQ ID NO:17 encode wild-type TasA and InhA1, respectively. Such a deletion also deletes the genes encoding regulatory proteins SinR and SinI.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysP1 protease, and a genetic modification that inactivates VpR protease.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; (e) the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; (f) the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; (g) the inactivated CysP1 protease is encoded by a genetically modified cysP1 gene at locus GBAA_1995 of the B. anthracis; and (h) the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA_34584 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the inactivated InhA2 protease is encoded by an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the inactivated TasA protease is encoded by a tasA gene that encodes an inactivated TasA comprising amino acid sequence SEQ ID NO:10; (d) the inactivated camelysin protease is encoded by a calY gene that encodes an inactivated camelysin comprising amino acid sequence SEQ ID NO:14; (e) the inactivated InhA1 protease is encoded by an inhA1 gene that encodes an inactivated InhA1 comprising amino acid sequence SEQ ID NO:18; (f) the inactivated MmpZ protease is encoded by a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22; (g) the inactivated CysP1 protease is encoded by a cysP1 gene that encodes an inactivated CysP1 comprising amino acid sequence SEQ ID NO:72; and (h) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR comprising amino acid sequence SEQ ID NO:76. In one embodiment, the amino acid sequence of the encoded inactivated NprB protease is SEQ ID NO:2. In one embodiment, the amino acid sequence of the encoded inactivated InhA2 protease is SEQ ID NO:6. In one embodiment, the amino acid sequence of the encoded inactivated TasA protease is SEQ ID NO:10. In one embodiment, the amino acid sequence of the encoded inactivated camelysin protease is SEQ ID NO:14. In one embodiment, the amino acid sequence of the encoded inactivated InhA1 protease is SEQ ID NO:18. In one embodiment, the amino acid sequence of the encoded inactivated MmpZ protease is SEQ ID NO:22. In one embodiment, the amino acid sequence of the encoded inactivated CysP1 protease is SEQ ID NO:72. In one embodiment, the amino acid sequence of the encoded inactivated VpR protease is SEQ ID NO:76.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhA1 protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17 (such a deletion also deletes genes encoding SinR and SinI); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22; (e) the inactivated CysP1 protease is encoded by a cysP1 gene that encodes an inactivated CysP1 comprising amino acid sequence SEQ ID NO:72; and (f) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR comprising amino acid sequence SEQ ID NO:76.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB consisting of amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 consisting of amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhA1 protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO:17 (such a deletion also deletes genes encoding SinR and SinI); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ consisting of amino acid sequence SEQ ID NO:22; (e) the inactivated CysP1 protease is encoded by a cysP1 gene that encodes an inactivated CysP1 consisting of amino acid sequence SEQ ID NO:72; and (f) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR consisting of amino acid sequence SEQ ID NO:76.

In some embodiments, a B. anthracis of the disclosure also includes a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. B. anthracis NprC is a neutral metalloprotease encoded by nprC, located at gene locus GBAA_2183 of B. anthracis “Ames ancestor” strain chromosome (GenBank Accession No. NC 003997). B. anthracis SprA is a serine protease encoded by sprA, located at gene locus GBAA_5414 of B. anthracis “Ames ancestor” strain chromosome. B. anthracis HtrA is a serine protease encoded by htrA, located at gene locus GBAA_3660 of B. anthracis “Ames ancestor” strain chromosome. HsIV protease is ATP-dependent protease HsIV that has been found in BH480 secrotome and is encoded at gene locus GBAA_3968 of B. anthracis “Ames ancestor” strain chromosome. Protease HslV and the ATPase/chaperone HslU are part of an ATP-dependent proteolytic system that is the prokaryotic homolog of the proteasome. HslV is a dimer of hexamers (a dodecamer) that forms a central proteolytic chamber with active sites on the interior walls of the cavity. HslV shares significant sequence and structural similarity with the proteasomal beta-subunit and both are members of the Ntn-family of hydrolases. HslV has a nucleophilic threonine residue at its N-terminus that is exposed after processing of the propeptide and is directly involved in active site catalysis.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases, a genetic modification that inactivates a protease of the transglutaminase-like superfamily, a genetic modification that inactivates a protease of peptidase family S8, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

The disclosure provides a B. anthracis comprising genetic modification that inactivates NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases that also includes a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhA1 protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysP1 protease, a genetic modification that inactivates VpR protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases also include genetic modifications that inactivate regulatory protein SinR, regulatory protein SinI, or both SinR and SinI. SinR and SinI are encoded by genes located at GBAA_1292 and GBAA_1293, respectively, of the B. anthracis genome.

Additional secreted proteases, the inactivation of which would further improve protein production by B. anthracis of the embodiments, can be identified using techniques known to those skilled in the art and as described herein.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases are sporulation-deficient. That is, any of the B. anthracis described herein can also comprise a genetic modification that prevents sporulation, such as a genetic modification that inactivates the Spo0A protein. The Spo0A protein, a key regulator of sporulation in Bacillus (see, e.g., Molle V et al., 2003, Molec. Microbiol. 50, 1683-1701), is encoded by the spo0A gene, located at locus GBAA_4394 of B. anthracis. B. anthracis that are unable to sporulate are advantageous as production organisms.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases are deficient in one or more virulence plasmids. One embodiment is a B. anthracis of the embodiments that is pXO1−, i.e., the B. anthracis lacks virulence plasmid pXO1. One embodiment is a B. anthracis of the embodiments that is pXO1−, pXO2−; i.e., the B. anthracis lacks virulence plasmids pXO1 and pXO2. One embodiment is a B. anthracis of the embodiments that is pXO1−, pXO2+.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA_1290 of the B. anthracis; a genetic modification that inactivates InhA1 protease, wherein the inactivated InhA1 protease is encoded by a genetically modified inhA1 gene at locus GBAA_1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA_3159 of the B. anthracis; a genetic modification that inactivates CysP1 protease, wherein the inactivated CysP1 protease is encoded by a genetically modified cysP1 gene at locus GBAA_1995 of the B. anthracis; a genetic modification that inactivates VpR protease, wherein the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA_34584 of the B. anthracis, and a genetic modification that inactivates Spo0A protein, wherein the inactivated Spo0A protein is encoded by a genetically modified spo0A gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXO1 and pXO2.

The disclosure provides a Bacillus anthracis having the identifying characteristics of B. anthracis BH480, deposited under ATCC Accession No. PTA-13162. A deposit of Bacillus anthracis BH480 has been made on Jul. 31, 2012 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, under ATCC accession number PTA-13162. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. Identifying characteristics of BH480 include (a) inactivated NprB, InhA2, TasA, camelysin, InhA1, MmpZ, CysP1, and VpR proteases, (b) sporulation-deficiency, and (c) lack of virulence plasmids pXO1 and pXO2. One embodiment of the disclosure is Bacillus anthracis BH480.

B. anthracis lacking more than one secreted protease can be produced using the methods disclosed herein. The disclosure provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases; effecting a genetic modification to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases; effecting a genetic modification to inactivate a protease of the transglutaminase-like superfamily; and effecting a genetic modification to inactivate a protease of peptidase family S8. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases and to inactivate a protease of the M6 family of metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, and to inactivate a protease of the M73 family of metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, and to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases, to inactivate a protease of the transglutaminase-like superfamily, and to inactivate a protease of peptidase family S8. Genetic modifications taught herein as well as those known to those skilled in the art can be used to inactivate proteases specified herein. Sporulation-deficient B. anthracis can be produced using techniques as taught herein or other techniques known to those skilled in the art. Those skilled in the art can also produce B. anthracis free of one or both virulence plasmids.

The disclosure provides a method to produce B. anthracis of the embodiments comprising: culturing the B. anthracis in a medium; and recovering the B. anthracis. Any suitable medium and culture conditions known to those skilled in the art can be used to produce such B. anthracis. Non-limiting examples of media include LB and FA, the compositions of which are specified in the Examples. Methods to recover the bacteria are also known to those skilled in the art.

The disclosure also provides a method to produce an endogenous B. anthracis protein, such as an endogenous secreted protein. Such a method comprises culturing a B. anthracis of the embodiments in a medium; and recovering the protein. In one embodiment, the protein is an endogenous secreted protein of B. anthracis. Culturing methods, recovery methods, and media to use are known to those skilled in the art.

The disclosure provides a modified Bacillus anthracis (B. anthracis) transformed with a recombinant molecule encoding a product, wherein the B. anthracis comprises any of the genetically modified B. anthracis disclosed herein (i.e., any B. anthracis of the embodiments), and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a protease of peptidase family S8; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector.

As used herein, a recombinant molecule comprises an expression vector operatively linked to a nucleic acid molecule encoding a desired product. A modified B. anthracis can comprise one or more recombinant molecules. A recombinant molecule can comprise one or more nucleic acid molecules encoding one or more desired products. As used herein, the phrase operatively linked means that the nucleic acid molecule encoding a desired product is joined to the vector in such a way that the product is produced. An expression vector is any vector that can transform B. anthracis (i.e., deliver a nucleic acid molecule encoding a product into B. anthracis) and that comprises expression control sequences that can effect expression in B. anthracis of the nucleic acid molecule encoding a desired product. Non-limiting examples of expression vectors are plasmid expression vectors, viral expression vectors, and other vectors known to those skilled in the art. Such a vector can be DNA, RNA, or a derivative of DNA or RNA. Examples of expression control sequences include, but are not limited to, a promoter, an enhancer, a repressor, a ribosome binding site, an RNA splice site, a polyadenylation site, a transcriptional terminator sequence, and a microRNA binding site. Examples of promoters include, but are not limited to, a promoter that controls expression of the pag gene that encodes B. anthracis protective antigen (PA), referred to herein as a pag promoter or PA promoter, and other promoters that function in B. anthracis. In one embodiment, the promoter is a pag promoter. A recombinant molecule can also comprise replication control sequences that can effect vector replication, such as an origin of replication. Selection of replication and expression control sequences to include can be accomplished by one skilled in the art. One embodiment is a recombinant molecule that is heterologous to B. anthracis; i.e., at least a portion of the recombinant molecule is not a natural B. anthracis plasmid. One embodiment is a recombinant molecule comprising a B. anthracis plasmid that is attenuated, for example, a virulence plasmid from which at least one component that would lead to virulence has been removed.

One embodiment is recombinant molecule pSJ136EFOS. One embodiment is a recombinant molecule comprising nucleic acid sequence SEQ ID NO:62.

One embodiment is a recombinant molecule selected from the group consisting of a recombinant molecule comprising nucleic acid sequence SEQ ID NO:65, a recombinant molecule comprising nucleic acid sequence SEQ ID NO:67, and a recombinant molecule comprising nucleic acid sequence SEQ ID NO:69. One embodiment is a recombinant molecule selected from the group consisting of pSW4-HBL L1 His, pSW4-HBL L2 His, and pSW4-HBL B His. One embodiment is recombinant molecule pSW4-HBL L1 His. One embodiment is recombinant molecule pSW4-HBL L2 His. One embodiment is recombinant molecule pSW4-HBL B His.

One embodiment is a recombinant molecule comprising a pSJ115 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:91. One embodiment is a recombinant molecule comprising a pYS5 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:92. One embodiment is a recombinant molecule selected from the group consisting of recombinant molecule pSJ136EF-His, recombinant molecule pSJ136EF-Cys, and recombinant molecule pSJ136EF-NEHY. One embodiment is recombinant molecule pSJ136EF-His. One embodiment is recombinant molecule pSJ136EF-Cys. One embodiment is recombinant molecule pSJ136EF-NEHY.

The disclosure also provides a protein comprising an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO:93, amino acid sequence SEQ ID NO:94, and amino acid sequence SEQ ID NO:95. One embodiment is a protein comprising amino acid sequence SEQ ID NO:93. One embodiment is a protein comprising amino acid sequence SEQ ID NO:94. One embodiment is a protein comprising amino acid sequence SEQ ID NO:95.

Methods to transform B. anthracis of the embodiments and to select and produce expression vectors and recombinant molecules with which to transform B. anthracis are described in the Examples, and can also be found, for example, in Sambrook J et al., 2001, Molecular Cloning: a Laboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press, and Ausubel F et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons.

A nucleic acid molecule encoding a desired product of the embodiments can encode a product that B. anthracis produces naturally (e.g., an anthrax toxin protein) or a product heterologous to B. anthracis (e.g., a toxin from a different organism). A heterologous product can be a combination of an endogenous and non-endogenous product (e.g., a tumor-targeting anthrax toxin). Examples of products include, but are not limited to, a protein, an amino acid, a nucleic acid molecule, a compound produced via a recombinant biosynthetic pathway, a small molecule, a drug, a vitamin, a drug conjugate, and a peptide nucleic acid conjugate.

One embodiment is a product comprising a toxin. Examples of toxins include, but are not limited to, an anthrax toxin, a cholera toxin, a diphtheria toxin, a hemolysin, and a ricin. Additional examples include, but are not limited to a Pseudomonas toxin, a Haemophilus ducreyi toxin, an Escherichia coli toxin, and a ribosome-inactivating protein (RIP) toxin. One embodiment is a product selected from the group consisting of an anthrax edema factor (EF), an anthrax lethal factor (LF), an anthrax protective antigen (PA), and combinations thereof. One embodiment is a product comprising an anthrax edema factor. One embodiment is a product comprising intact anthrax edema factor. One embodiment is a product comprising an anthrax lethal factor. One embodiment is a product comprising intact anthrax lethal factor. One embodiment is a product comprising an anthrax protective antigen. One embodiment is a product comprising intact anthrax protective antigen. One embodiment is a product comprising an anthrolysin. One embodiment is a product comprising intact anthrolysin. One embodiment is a product comprising a hemolysin. One embodiment is a product comprising a Bacillus cereus hemolysin HBL. One embodiment is a product selected from the group consisting of a B. cereus hemolysin HBL L1, a B. cereus hemolysin HBL L2, a B. cereus hemolysin HBL B, and combinations thereof. One embodiment is a product comprising a diphtheria toxin. One embodiment is a product comprising a cholera toxin. One embodiment is a product comprising a ricin. One embodiment is a product comprising a Pseudomonas toxin. One embodiment is a product comprising a Haemophilus ducreyi toxin. One embodiment is a product comprising an Escherichia coli toxin. One embodiment is a product comprising a ribosome-inactivating protein (RIP) toxin.

One embodiment is a product comprising a toxin fusion protein. One embodiment is a product comprising a toxin conjugated to a tumor target. Examples of tumor targets are known to those skilled in the art. Examples of conjugates include, but are not limited to, an anthrax toxin conjugated to a tumor target, a cholera toxin conjugated to a tumor target, a diphtheria toxin conjugated to a tumor target, a hemolysin conjugated to a tumor target, and a ricin conjugated to a tumor target. One embodiment is an anthrax toxin conjugated to a tumor target. One embodiment is a product comprising a fusion protein comprising a PA binding moiety located in the amino terminal region of EF or LF (e.g., the amino terminal about 250 to about 260 amino acids of the mature protein) joined to a protein of interest that can be delivered to a cell via the protective antigen (PA) cellular translocation mechanism known to those skilled in the art.

A nucleic acid molecule of the embodiments can encode a natural product or any variant thereof that retains the activity of the natural product. Nucleic acid molecules of the embodiments can be produced using a number of methods known to those skilled in the art; see, for example, Sambrook J et al., ibid. and Ausubel F et al., ibid. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecules of the embodiments can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., EF expressed from a nucleic acid molecule that encodes an EF protein can be tested for its activity in a potency assay, such as that described in the Examples).

Products of the embodiments can be produced by culturing modified B. anthracis transformed with a recombinant molecule encoding a product of the embodiments. The disclosure provides a method to produce a product comprising: culturing a modified B. anthracis transformed with a recombinant molecule encoding a product of the embodiments; and recovering the product. Methods to effect such production, including culturing such modified B. anthracis and recovering such products are described in the Examples and are known to those skilled in the art, see for example Sambrook J et al., ibid, and Ausubel, F et al., ibid.

The disclosure provides antibodies against products produced by B. anthracis of the embodiments. Examples of antibodies include polyclonal antibodies, monoclonal antibodies, and functional equivalents such as antibody fragments and genetically-engineered antibodies (including, but not limited to, single chain antibodies and chimeric antibodies that can bind to more than one epitope) that retain antibody binding activity. One embodiment is an antibody against an anthrax edema factor produced by a modified B. anthracis of the embodiments.

The disclosure provides methods to use products of the embodiments. The disclosure provides that antibodies produced against products produced by modified B. anthracis of the embodiments can be used to detect such products or, as appropriate, organisms or toxins corresponding to such products. One embodiment is a method to determine if a sample comprises anthrax toxin, the method comprising: contacting a sample with an antibody against an anthrax edema factor produced by a modified B. anthracis of the embodiments; and determining whether the antibody forms a complex, wherein detection of a complex indicates that the sample comprises anthrax. One embodiment is a method to determine if a sample comprises anthrax toxin, the method comprising: contacting a sample with an antibody against intact anthrax edema factor produced by a B. anthracis of the embodiments; and determining whether the antibody forms a complex, wherein detection of a complex indicates that the sample comprises anthrax. Methods to accomplish such detection are known to those skilled in the art.

The disclosure provides that products produced by modified B. anthracis of the embodiments can be used for their intended purpose. For example, products of agents that cause disease can be used to protect (e.g., prevent or treat) an animal from such disease. One embodiment is a method to protect an animal from anthrax, the method comprising administering an anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. One embodiment is a method to protect an animal from anthrax, the method comprising administering intact anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. Such an anthrax edema factor can be administered alone or in combination with one or more other agents, such as with an anthrax lethal factor and/or protective antigen. One embodiment is a method to protect an animal from anthrax, the method comprising administering an antibody against intact anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. Such an anti-anthrax edema factor can be administered alone or in combination with one or more other agents, such as with an antibody against an anthrax lethal factor and/or protective antigen. Methods to accomplish such administration are known to those skilled in the art.

The following is a listing of the SEQ ID NOs disclosed in the application. It is to be appreciated that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the embodiments and apparent amino acid sequences of proteins of the embodiments.

SEQ ID NO: Species Description 1 B. anthracis Wild-type NprB protease (full-length encoded protein  (i.e., includes signal sequence)); note that the mature  NprB protease begins at amino acid 28 of SEQ ID NO: 1 2 Synthetic Inactivated NprB protease (full-length encoded protein) 3 Synthetic Portion of nprB nucleic acid comprising loxP insert 4 Synthetic Translation of SEQ ID NO: 3 5 B. anthracis Wild-type InhA2 protease (full-length encoded protein);  note that the mature InhA2 protease begins at amino acid  33 of SEQ ID NO: 5 6 Synthetic Inactivated InhA2 protease (full-length encoded protein) 7 Synthetic Portion of inhA2 nucleic acid comprising loxP insert 8 Synthetic Translation of SEQ ID NO: 7 9 B. anthracis Wild-type TasA protease (full-length encoded protein); note that the mature TasA protease begins at amino acid  30 of SEQ ID NO: 9 10 Synthetic Inactivated TasA protease (full-length encoded protein) 11 Synthetic Portion of tasA nucleic acid comprising loxP insert 12 Synthetic Translation of SEQ ID NO: 11 13 B. anthracis Wild-type camelysin protease (full-length encoded protein); note that the mature camelysin protease begins at amino acid 30 of SEQ ID NO: 13 14 Synthetic Inactivated camelysin protease (full-length encoded protein) 15 Synthetic Portion of calY nucleic acid comprising loxP insert 16 Synthetic Translation of SEQ ID NO: 15 17 B. anthracis Wild-type InhA1 protein (full-length encoded protein); note that the mature InhA1 protease begins at amino acid 32 of SEQ ID NO: 17 18 Synthetic Inactivated InhA1 protease (full-length encoded protein) 19 Synthetic Portion of inhA1 nucleic acid comprising loxP insert 20 Synthetic Translation of SEQ ID NO: 19 21 B. anthracis Wild-type MmpZ protein (full-length encoded protein); note that the mature MmpZ protease begins at amino acid 27 of SEQ ID NO: 21 22 Synthetic Inactivated MmpZ protease (full-length encoded protein) 23 Synthetic Portion of mmpZ nucleic acid comprising loxP insert 24 Synthetic Translation of SEQ ID NO: 23 25 Synthetic Inactive TasA-InhA1 translation product 26 Synthetic Portion of tasA-inhA1 nucleic acid comprising loxP insert 27 Synthetic Translation of SEQ ID NO: 26 28 B. anthracis Wild-type BA1995 protein (full-length encoded protein) 29 B. anthracis Wild-type VpR protein (full-length encoded protein) 30 B. anthracis Wild-type BA5414 protein (full-length encoded protein) 31 Synthetic Zinc-binding motif for zincin tribe of metalloproteases: His-Glu-Xxx-Xxx-His 32 Synthetic Extended zinc-binding motif of metzincin metalloproteases: His-Glu-Xxx-Xxx-His-Xxx-Xxx-Gly/Asn-Xxx-Xxx-His/Asp 33 B. anthracis IMSGGSWAGKIAGTTPTSFS 34 Synthetic Primer 0672LL: GCTCGAGCGGATGTACATCTGTAATGAGT 35 Synthetic Primer 0672LR: GGATATCTTGAACGATGTGACCAAATG 36 Synthetic Primer 0672RL: GCCCGGGCCTGTCGAAGCTTGGTCATT 37 Synthetic Primer 0672RR: GCCGCGGTTTGCATACCTGTGTTACCG 38 Synthetic Primer 0672seqF: GGTCAAGAAGCTGGTGGAGGTA 39 Synthetic Primer 0672seqR: TCTGTTCCTGCAATTTTCCC 40 Synthetic Primer 1288LL: GCTCGAGTAATTTGGAAGGTGATTAGC 41 Synthetic Primer 1288LR: GCCCGGGTTCACATCTTCTACATTGTAAT 42 Synthetic Primer 1288RL: GACTAGTAACAATCGTAAAAGAAACAGCG 43 Synthetic Primer 1288RR: GGAGCTCTATCGATCGCCTGTAAATTC 44 Synthetic Primer 1288seqF: GGGGATATGGACATGACTTT 45 Synthetic Primer 1288seqR: CAGTAAGTGTGTCACCCTTC 46 Synthetic Primer 1290L: GAGAAGATAGCTGCTGAGAG 47 Synthetic Primer 1290R: TAGAGGGAGTTTAATGGGGA 48 Synthetic Primer 1290seqF: GAAATTGCGCAAAAAGAT 49 Synthetic Primer 1290seqR: AGAGCCATTCCAGAACGC 50 Synthetic Primer 3159LL: GCTCGAGGGGTAATAACTTTCAATTAATAC 51 Synthetic Primer 3159LR: GGATATCGAAAAAACAAACACAGTACC 52 Synthetic Primer 3159RL: GCCCGGGGGTTGGCAAGCTGCCGATTC 53 Synthetic Primer 3159RR: GCCGCGGCGAATGGTTCAATTGCTCCG 54 Synthetic Primer 3159seqF: GGTACTGTGTTTGTTTTTTC 55 Synthetic Primer 3159seqR: GAATCGGCAGCTTGCCAACC 56 Synthetic Primer 3159CF: CCTCGAGTTTCATTTTTGAAGTCTTCTTC 57 Synthetic Primer 3159CR: CACTAGTCAGCGAAACGATGATTGATTTT 58 Synthetic Primer deltaF: TCCGATTAGGAAGTTGACAA 59 Synthetic Primer deltaR: CAGTTACCACCAATTGTTTT 60 Synthetic Primer deltaCF: CCTCGAGTTTTCTTATTGCATTTCTAATGTG 61 Synthetic Primer deltaCR: CCCGCGGTTAGCGATATAAGCGAACAG 62 Synthetic pSJ136EFOS 63 Synthetic Translation of EF from SEQ ID NO: 62 64 Synthetic Translation of Mature EF from SEQ ID NO: 62 65 Synthetic pSW4-HBL L1 His 66 Synthetic Translation of HBL L1 from SEQ ID NO: 65 67 Synthetic pSW4-HBL L2 His 68 Synthetic Translation of HBL L2 from SEQ ID NO: 67 69 Synthetic pSW4-HBL B His 70 Synthetic Translation of HBL B His from SEQ ID NO: 69 71 B. anthracis Wild-type CysP1 protein (full-length encoded protein); note that the mature CysP1 protease begins at amino acid 31 of SEQ ID NO: 71 72 Synthetic Inactivated CysP1 protease (full-length encoded protein) 73 Synthetic Portion of cysP1 nucleic acid comprising FRT insert 74 Synthetic Translation of SEQ ID NO: 73 75 B. anthracis Wild-type VpR protein (full-length encoded protein); note that the mature VpR protease begins at amino acid 26 of SEQ ID NO: 75 76 Synthetic Inactivated VpR protease (full-length encoded protein) 77 Synthetic Portion of vpR nucleic acid comprising FRT insert 78 Synthetic Translation of SEQ ID NO: 77 79 Synthetic Primer 1995LL: ACTGCTCGAGTGGGCTGACACATTTAAAAG 80 Synthetic Primer 1995LR: ACTGACTAGTAGTTGAACAAAGTGCGGCAG 81 Synthetic Primer 1995RL: ACTGCTCGAGAATGAAATAAACTGGCCAAAAGGTG 82 Synthetic Primer 1995RR: ACTGACTAGTCGGGAAAAACTTCAAATCCA 83 Synthetic Primer 1995 seqF: TTGCCAGAGCTTTTCATTGA 84 Synthetic Primer 1995 seqR: CGCTAATGAATAATCTGCCA 85 Synthetic Primer 4584LL: ACTGCTCGAGAAGCTGTCGGTACTGCTAAA 86 Synthetic Primer 4584LR: ACTGACTAGTCGAGTGCCATACTTAAAAGTATAGA 87 Synthetic Primer 4584RL: ACTGCTCGAGATCCTTGGGAGAAAAATTACGGCATT 88 Synthetic Primer 4584RR: ACTGACTAGTCGCCAAACATTCATTCATTTCTTCT 89 Synthetic Primer 4584 seqF: TGAGTGAAACGGCGTAACTT 90 Synthetic Primer 4584 seqR: TATTCCTTCAAAGCCGATAT 91 Synthetic LFn-B1aY protein 92 Synthetic PA SNKE dFF E308D protein 93 Synthetic EF-His protein 94 Synthetic EF-Cys protein 95 Synthetic EF-NEHY protein

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

Example 1 Materials and Methods

Materials

Proteases and genes encoding such proteases that were used and/or analyzed herein are listed in Table 1.

TABLE 1 B. anthracis Ames ancestor strain genes and proteins inactivated and/or analyzed herein. Protein Gene Function/Name Locus Tag NprB nprB Metallopeptidase GBAA_0599 InhA2 inhA2 Metallopeptidase GBAA_0672 TasA tasA Metallopeptidase GBAA_1288 Camelysin calY Metallopeptidase GBAA_1290 SinR sinR regulatory protein GBAA_1292 SinI sinI regulatory protein GBAA_1293 InhA1 inhA1 Metallopeptidase GBAA_1295 MmpZ mmpZ Metallopeptidase GBAA_3159 CysP1 cysP1 putative cysteine protease GBAA_1995 VpR vpR minor extracellular protease GBAA_4584 SprA sprA serine protease GBAA_5414 NprC nprC neutral metalloprotease GBAA_2183 HtrA htrA serine protease GBAA_3660 HsIV hsIV Prokaryotic homolog of GBAA_3968 proteasome HsIV ALO alo thiol-activated cytolysin GBAA_3355 (anthrolysin) Spo0A spo0A sporulation regulator GBAA_4394 EF cya edema factor GBAA_pXO1_0142 PA pag protective antigen GBAA_pXO1_0164 LF lef lethal factor GBAA_pXO1_0172

Oligonucleotide primers produced and/or used herein are listed in Table 2.

TABLE 2 Primers produced and/or used herein. Primer Sequence ^(a) (5′-3′)(location) Relevant property Site 0672LL GCTCGAGCGGATGTACATCTGTAATGAGT Primer pair to amplify left XhoI (SEQ ID NO: 34) fragment of inhA2gene to clone 0672LR GGATATCTTGAACGATGTGACCAAATG it into pDC EcoRV (SEQ ID NO: 35) 0672RL GCCCGGGCCTGTCGAAGCTTGGTCATT Primer pair to amplify right  SmaI (SEQ ID NO: 36) fragment of inhA2 gene to clone 0672RR GCCGCGGTTTGCATACCTGTGTTACCG it into pDC SacII (SEQ ID NO: 37) 0672seqF GGTCAAGAAGCTGGTGGAGGTA Primer pair to verify inhA2gene (SEQ ID NO: 38) disruption 0672seqR TCTGTTCCTGCAATTTTCCC (SEQ ID NO: 39) 1288LL GCTCGAGTAATTTGGAAGGTGATTAGC Primer pair to amplify left XhoI (SEQ ID NO: 40) fragment of tasA gene to clone it 1288LR GCCCGGGTTCACATCTTCTACATTGTAAT into pSC SmaI (SEQ ID NO: 41) 1288RL GACTAGTAACAATCGTAAAAGAAACAGCG Primer pair to amplify right SpeI (SEQ ID NO: 42) fragment of tasA gene to clone it 1288RR GGAGCTCTATCGATCGCCTGTAAATTC into pSC SacI (SEQ ID NO: 43) 1288seqF GGGGATATGGACATGACTTT (SEQ ID NO: 44) Primer pair to verify tasA gene 1288seqR CAGTAAGTGTGTCACCCTTC (SEQ ID NO: 45) disruption 1290L GAGAAGATAGCTGCTGAGAG Primer pair to amplify calY gene (SEQ ID NO: 46) to clone it into pDC 1290R TAGAGGGAGTTTAATGGGGA (SEQ ID NO: 47) 1290seqF GAAATTGCGCAAAAAGAT (SEQ ID NO: 48) Primer pair to verify calY gene 1290seqR AGAGCCATTCCAGAACGC (SEQ ID NO: 49) disruption 3159LL GCTCGAGGGGTAATAACTTTCAATTAATAC Primer pair to amplify left XhoI (SEQ ID NO: 50) fragment of mmpZ gene to clone 3159LR GGATATCGAAAAAACAAACACAGTACC it into pSC EcoRV (SEQ ID NO: 51) 3159RL GCCCGGGGGTTGGCAAGCTGCCGATTC Primer pair to amplify right SmaI (SEQ ID NO: 52) fragment of mmpZ gene to clone 3159RR GCCGCGGCGAATGGTTCAATTGCTCCG it into pSC SacII (SEQ ID NO: 53) 3159seqF GGTACTGTGTTTGTTTTTTC  Primer pair to verify mmpZ gene (SEQ ID NO: 54) disruption 3159seqR GAATCGGCAGCTTGCCAACC (SEQ ID NO: 55) 3159CF CCTCGAGTTTCATTTTTGAAGTCTTCTTC Primer pair to complement XhoI (SEQ ID NO: 56) mmpZ gene disruption 3159CR CACTAGTCAGCGAAACGATGATTGATTTT SpeI (SEQ ID NO: 57) 1995LL ACTGCTCGAGTGGGCTGACACATTTAAAAG Primer pair to amplify left XhoI (SEQ ID NO: 79) fragment of cysP1 gene to 1995LR ACTGACTAGTAGTTGAACAAAGTGCGGCAG clone it into pSCF SpeI (SEQ ID NO: 80) 1995RL ACTGCTCGAGAATGAAATAAACTGGCCAAA Primer pair to amplify right XhoI (SEQ ID NO: 81) fragment of cysP1 gene to 1995RR ACTGACTAGTCGGGAAAAACTTCAAATCCA clone it into pSCF SpeI (SEQ ID NO: 82) 1995 TTGCCAGAGCTTTTCATTGA Primer pair to verify cysP1 seqF (SEQ ID NO: 83) gene disruption 1995 CGCTAATGAATAATCTGCCA seqR (SEQ ID NO: 84) 4584LL ACTGCTCGAGAAGCTGTCGGTACTGCTAAA Primer pair to amplify left XhoI (SEQ ID NO: 85) fragment of vpR gene to clone 4584LR ACTGACTAGTCGAGTGCCATACTTAAAAGT it into pSCF SpeI ATAGA (SEQ ID NO: 86) 4584RL ACTGCTCGAGATCCTTGGGAGAAAAATTAC Primer pair to amplify right XhoI GGCATT (SEQ ID NO: 87) fragment of vpR gene to clone 4584RR ACTGACTAGTCGCCAAACATTCATTCATTTC it into pSCF SpeI TTCT (SEQ ID NO: 88) 4584 TGAGTGAAACGGCGTAACTT Primer pair to verify vpR gene seqF (SEQ ID NO: 89) disruption 4584 TATTCCTTCAAAGCCGATAT seqR (SEQ ID NO: 90) deltaF TCCGATTAGGAAGTTGACAA Primer pair to verify deletion of (SEQ ID NO: 58) tasA-inhAl region deltaR CAGTTACCACCAATTGTTTT (SEQ ID NO: 59) deltaCF CCTCGAGTTTTCTTATTGCATTTCTAATGTG Primer pair to complement tasA- XhoI TTCG (SEQ ID NO: 60) inhAl region deletion deltaCR CCCGCGGTTAGCGATATAAGCGAACAG SacII (SEQ ID NO: 61) ^(a) Restriction site is underlined

Plasmids produced, used, and/or analyzed herein are listed in Table 3.

TABLE 3 Plasmids produced and/or used herein. Plasmid Relevant characteristic(s) pHY304 Contains Em^(R) gene and strongly temperature-sensitive replicon for both E. coli and gram-positive bacteria; Em^(R) in both E. coli and B. anthracis pDC Ω-sp cassette flanked by two similarly oriented loxP sites and two external multiple restriction sites (single XhoI, SalI, and EcoRV upstream of first loxP and single PstI, XmaI and SacII downstream of second loxP) inserted into pHY304 pSC Plasmid used for single crossovers in B. anthracis. Ap^(R) in E. coli; Em^(R) both in E. coli and B. anthracis pSCF Plasmid used for single crossovers in B. anthracis. Ap^(R) in E. coli; Em^(R) both in E. coli and B. anthracis. Two FRT sites flank multiple restriction sites area. pInhA2I pDC with loxP-Ω-sp-loxP flanked 3′ and 5′ by inhA2 gene sequences pTasALI pSC containing tasA fragment amplified with primer pair 1288LL/1288LR pTasARI pSC containing tasA fragment amplified with primer pair 1288RL/1288RR pCamI pHY304 with loxP-Ω-sp-loxP flanked 3′ and 5′ by calY gene sequences pMmpZLI pSC containing mmpZ fragment amplified with primer pair 3159LL/3159LR pMmpZRI pSC containing mmpZ fragment amplified with primer pair 3159RL/3159RR pCysP1LI pSCF containing cysP1 fragment amplified with primer pair 1995LL/1993LR pCysP1RI pSCF containing cysP1 fragment amplified with primer pair 1995RL/1995RR pVpRLI pSCF containing vpR fragment amplified with primer pair 4584LL/4584LR pVpRRI pSCF containing vpR fragment amplified with primer pair 4584RL/4584RR pCrePA Contains cre gene and strongly temperature-sensitive replicon for both E. coli and gram-positive bacteria; Em^(R) in both E. coli and B. anthracis pCrePAS Contains cre gene and strongly temperature-sensitive replicon for both E. coli and gram-positive bacteria; Sp^(R) in both E. coli and B. anthracis pFPAS Contains flp gene and strongly temperature-sensitive replicon for both E. coli and gram-positive bacteria; Sp^(R) in both E. coli and B. anthracis pMmpZC pSC with 3159F/3159R PCR fragment containing entire mmpZ gene pΔTasA- pSC with deltaCF/deltaCR fragment containing entire InhA1C tasA-inhA1 DNA region pSΩL304 pDC with loxP-Ω-sp-loxP flanked 3′ and 5′ by spo0A sequences pSJ136EFOS Contains B. anthracis cya gene instead of the lef gene in pSJ115 (Park S et al., 2000, Protein Expr. Purif. 18, 293-302. pSW4-HBL L1 Encodes B. cereus HBL L1 His His pSW4-HBL L2 Encodes B. cereus HBL L2 His His pSW4-HBL B Encodes B. cereus HBL B His His

Bacterial strains produced, used, and/or analyzed herein are listed in Table 4.

TABLE 4 Bacterial strains produced and/or used herein. Strain Relevant characteristic(s) A33 B. anthracis Ames 33 strain (pXO1⁻ pXO2⁻) A35 B. anthracis Ames 35 strain (pXO1⁺ pXO2⁻) A35ΔSpo0A Spo0A knockout containing one loxP site, previous name of this strain is SΩL35 A35ΔNprB nprB knockout containing one loxP site A35ΔInhA2 inhA2 knockout containing one loxP site A35ΔTasA tasA knockout containing one loxP site A35ΔCam calY knockout containing one loxP site A35ΔInhA1 inhA1 knockout containing one loxP site A35ΔMmpZ mmpZ knockout containing one loxP site A35ΔmmpZC A35ΔMmpZ complemented in situ by insertion of native mmpZ gene A35DM Ames 35 double mutant with nprB and inhA1 knockouts; each gene contains one loxP site A35TM Ames 35 tetra-protease mutant; has nprB knockout containing one loxP site and deleted DNA region including tasA, calY, sinI, sinR, and inhA1 genes A35TMC A35TM with tasA-inhA1 deletion restored in situ by insertion of native DNA region including tasA, calY, sinI, sinR, and inhA1 genes A35PM Ames 35 penta-protease mutant; 35TM with inhA2 knockout containing one loxP site; total of 3 loxP sites in chromosome A35HM Ames 35 hexa-protease mutant: A35PM with mmpZ knockout containing one loxP site; total of 4 loxP sites in chromosome A35HMS A35HM with spo0A knockout containing one loxP site; total of 5 loxP sites in chromosome BH460 A35HMS cured of pXO1 BH480 BH460 with both cysP1 and vpR knockout containing FRT site in each knockout gene; total of 5 loxP and two FRT sites in chromosome BH450 A33; nprB and spo0A knockouts, residual loxP sites in both nprB and spo0A genes; strain was previously named MSLL33 Bacterial Growth Conditions and Phenotypic Characterization

E. coli strains were grown in Luria-Bertani (LB) broth and used as hosts for cloning. LB agar was used for selection of transformants (Sambrook J et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold. Spring Harbor, N.Y.). B. anthracis strains were also grown in LB or FA medium (Singh Y et al., 1989, J. Biol. Chem. 264, 19103-19107). FA medium contains, per liter, 33 g of tryptone, 20 g of yeast extract, 7.4 g of NaCl, 8 g of Na₂HPO₄, and 4 g of KH₂P0₄, pH 7.5. Antibiotics (Sigma-Aldrich, St. Louis, Mo.) were added to the medium when appropriate to give the following final concentrations: ampicillin (Ap), 100 μg/ml (only for E. coli); erythromycin (Em), 400 μg/ml for E. coli and 10 μg/ml for B. anthracis; spectinomycin (Sp), 150 μg/ml for both E. coli and B. anthracis; kanamycin (Km), 20 μg/ml (only for B. anthracis). SOC medium (Quality Biologicals, Inc., Gaithersburg, Md.) was used for outgrowth of transformation mixtures prior to plating on selective medium. B. anthracis spores were prepared from strains capable of sporulating as previously described (Thorne C, 1993, in Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics, 113-124, Sonenshein A B et al., (Eds.), American Society for Microbiology, Washington, D.C.) after growth on NBY minimal agar (nutrient broth, 8 g/liter; yeast extract, 3 g/liter; MnSO₄.H₂O, 25 mg/liter; agar, 15 g/liter) at 30° C. 5 days. Spores and vegetative cells were visualized with a Nikon Eclipse E600W light microscope (Nikon Instrument Inc., New York).

DNA Isolation and Manipulation

Preparation of plasmid DNA from E. coli, transformation of E. coli, and recombinant DNA techniques were carried out by standard procedures (Sambrook J et al., ibid.). E. coli SCS110 competent cells were purchased from Stratagene (La Jolla, Calif.), and E. coli TOP10 competent cells were purchased from Invitrogen (Carlsbad, Calif.). Recombinant plasmid construction was carried out in E. coli TOP10. Plasmid DNA from B. anthracis was isolated according to the protocol for the purification of plasmid DNA from B. subtilis (Qiagen, Valencia, Calif.). Chromosomal DNA from B. anthracis was isolated with the Wizard genomic purification kit (Promega, Madison, Wis.). B. anthracis was electroporated with unmethylated plasmid DNA isolated from E. coli SCS110 (dam⁻ dcm⁻). Electroporation-competent B. anthracis cells were prepared and transformed as previously described (Pomerantsev A P et al., 2009, J. Bacteriol. 191, 5134-5136). Restriction enzymes. T4 ligase, and Antarctic phosphatase were purchased from New England Biolabs (Ipswich, Mass.). Taq polymerase, Platinum PCR SuperMix High Fidelity kit and the TOPO TA cloning kit were from Invitrogen. The pGEM-T Easy Vector system was from Promega. Ready-To-Go PCR Beads were from GE Healthcare Biosciences Corp. (Piscataway, N.J.). For routine PCR analysis, a single colony was suspended in 200 μl of TE buffer (pH 8.0), heated to 95° C. for 45 s, and then cooled to room temperature (Sambrook J et al., ibid.). Cellular debris was removed by centrifugation at 15,000×g for 10 min. Two microliters of the lysate contained sufficient template to support PCR. The GeneRuler DNA Ladder Mix from MBI Fermentas (Glen Burnie, Md.) was used to assess DNA fragment length. All constructs were verified by DNA sequencing and/or restriction enzyme digestion.

Construction of Vectors for Protease Gene Inactivation

B. anthracis Ames 35 (pXO1+pXO2−) (A35) was used for genetic manipulations. The GenBank database (GenBank Accession No. for the Ames strain is NC_003997) was analyzed for the identification of target genes and for the corresponding primer design. The Cre/Lox genetic modification method was adapted to introduce precise genetic knockouts into B. anthracis genes encoding putative proteases. The general schemes for producing B. anthracis mutants using Cre-loxP system were described previously (Pomerantsev A P et al., 2006, ibid.; Pomerantsev A P et al., 2009, ibid.). The system employs vectors designated generically as pDC, for double-crossover plasmid or pSC, for single-crossover plasmid. These plasmids are derived from the highly temperature-sensitive plasmid pHY304 (Pritzlaff et al., 2001, Mol. Microbiol. 39, 236-247), which has permissive and restrictive temperatures of 30° C. and 37° C., respectively. The pDC plasmid (Pomerantsev A P et al., 2006, ibid.) was used to inactivate the spo0A (GBAA_4394), nprB (GBAA_0599) (Pomerantsev A P et al., 2006, ibid.), inhA1 (GBAA_1295) (Kastrup C J et al., ibid.), inhA2 (GBAA_0672), and calY (GBAA_1290) genes. The pSC plasmid (Pomerantsev A P et al., 2009, ibid.) was used to inactivate the tasA (GBAA_1288) and mmpZ (GBAA_3159) genes. Both plasmids were used in the production of a genomic deletion of the region from tasA (GBAA_1288) to inhA1 (GBAA_1295).

The nprB gene was inactivated as described in Pomerantsev et al., 2006, ibid. The inhA1 gene was inactivated as described in Kastrup C J et al., ibid. To inactivate the inhA2 gene, left and right fragments were amplified with primer pairs 0672LL/0672LR and 0672RL/0672RR, respectively, (Table 2) and inserted into pDC to produce the pInhA2I plasmid (I at the end of the proteases gene number means inactivation). To inactivate the tasA gene, left and right fragments were amplified with primer pairs 1288LL/1288LR and 1288RL/1288RR, respectively, and inserted into pSC to produce the pTasALI and pTasARi plasmids. To inactivate the camelysin gene calY, a DNA fragment overlapping the protease gene was amplified with primer pair 1290L/1290R and inserted into the EcoRI-site of pHY304. The internal BglII-fragment of the calY gene was replaced with a loxP-Ω-sp-loxP cassette flanked by two BglII sites (Pomerantsev A P et al., 2006, ibid.) to create the pCamI plasmid for the gene inactivation. To inactivate the mmpZ gene, left and right fragments were amplified with primer pairs 3159LL/3159LR and 3159RL/3159RR, respectively, and inserted into pSC to produce pMmpZLI and pMmpZRI plasmids. To delete the tasA-inhA1 gene cluster, the double protease mutant A35DM strain (having a LoxP site in the inhA1 gene) was transformed with pTasALI, and the plasmid was integrated into the genome as described previously (Pomerantsev A P et al., 2009, ibid.). Subsequent transformation of the recombinant strain with the pCrePAS plasmid (Pomerantsev A P et al., 2009, ibid.) eliminated the complete tasA-inhA1 gene cluster and produced the tetra-protease mutant strain A35TM.

The Crc recombinase-expressing plasmids pCrePAS (Pomerantsev A P et al., 2009, ibid.) and pCrePA (Pomerantsev A P et al., 2006, ibid.) both have permissive and restrictive temperatures of 30° C. and 37° C., respectively, and differ only in the selectable marker. The pCrePA was used for elimination of DNA regions containing a spectinomycin resistance cassette located between two similarly oriented loxP sites (Pomerantsev A P et al., 2006, ibid.), while pCrePAS was used in a similar way when the recipient strain did not contain a spectinomycin marker. In that case, the region to be deleted generally contained an erythromycin resistance gene along with backbone plasmid pSC (Pomerantsev A P et al., 2009, ibid.). In both cases, a single loxP site replaced the DNA region targeted for deletion.

To complement the mutation in the mmpZ gene (A35ΔMmpZ strain) and to restore the deleted taksA-inhA1 region in the A35TM strain, the 3159CF/3159CR PCR fragment (amplified using primer pair 3159CF/3159CR) and the deltaCF/deltaCR PCR fragment (amplified using primer pair delta CF/delta CR) were inserted into the pSC plasmid. The resulting pMmpZC and pΔTasA-InhA1C plasmids containing, respectively the intact mmpZ gene and the whole tasA-inhA1 region (C at the end of the proteases gene means complementation), were used for complementation. Each plasmid was inserted separately into the corresponding mutant by a single crossover event. During the crossover, both the mmpZ gene and whole tasA-inhA1 region were inserted into the genomes of the corresponding mutants. Subsequent elimination of the plasmid sequences by Cre recombinase left an intact functional copy of the originally mutated gene along with an inactive duplicate copy of a fragment of the gene.

Preparation of Protease-Deficient B. anthracis

B. anthracis strains A33 (Pomerantsev A P et al., 2003, Infect Immun 71, 6591-6606), A35, A35ΔSpo0A, A35ΔNprB, BH450 (the latter four all described in Pomerantsev A P et al., 2006, ibid.), and A35ΔInhA1 (Kastrup C J et al., ibid.) and their relevant characteristics are listed in Table 4. Also listed in Table 4 are protease-deficient B. anthracis strains produced as described herein. B. anthracis that was genetically modified to inactivate NprB, InhA2, TasA, camelysin. InhA1, or MmpZ, respectively, was constructed in the A35 strain by the replacement of the respective coding sequences with the loxP element as described herein.

The double NprB, InhA1 mutant (A35DM), a B. anthracis comprising a genetic modification to inactivate NprB and a genetic modification to inactivate InhA1, was created starting from the A35ΔNprB strain. The tetra-protease mutant, A35TM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate camelysin, and a genetic modification to inactivate InhA1) was created by deletion of the tasA-inhA1 gene region in the A35DM strain. The A35TM was then used for inactivation of the inhA2 and mmpZ genes with plasmids pInhA21 and pMmpZLI/pMmpZR1. The resulting penta- and hexa-protease mutants were designated A35PM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate camelysin, a genetic modification to inactivate InhA1, and a genetic modification to inactivate InhA2) and A35HM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate camelysin, a genetic modification to inactivate InhA1, a genetic modification to inactivate InhA2, and a genetic modification to inactivate MmpZ), respectively.

The mutant strains were checked at each step to ensure they had retained the ability to sporulate (Sastalla et al., 2010, Appl. Environ. Microbiol. 76, 6318-6321). To intentionally produce a sporulation-deficient hexa-protease mutant of A35HM, A35HMS was produced by inactivating the spo0A (GBAA_4394) gene in A35HM using the plasmid pSLΩL304 (Pomerantsev et al., 2006). The final protease-deficient spo0A-negative mutant lacking pXO1 was obtained by repeated passage of the A35HMS mutant at elevated temperatures to cure pXO1 as described previously (Pomerantsev et al., 2003, ibid.). The final strain was designated B. anthracis BH460 (Table 4).

PCR and Sequence Analysis of Chromosomal Modifications

PCR fragments containing loxP sites within mutated (i.e., inactivated) genes were amplified and sequenced using primers listed in Table 2 (0672seqF/0672seqR, 1288seqF/1288seqR, 1290seqF/1290seqR, 3159seqF/3159seqR). All primers for PCR and sequencing were synthesized by Operon Biotechnologies, Inc. (Huntsville, Ala.) or the FDA core facility (Bethesda, Md.). Sequences were determined from both sides of the PCR fragments (Macrogen, Rockville, Md.). For verification of the genomic deletion in the tasA-inhA1 gene area, the region encompassing the start of tasA gene and the end of inhA1 gene was amplified in the mutant strains by PCR using the primer pair deltaF/deltaR. The location of the loxP site inside the PCR fragments was determined by sequencing the fragments.

Western Blot Analysis

The A35 strain and genetically modified B. anthracis mutants were grown at 37° C. in LB in air to analyze B. anthracis toxin production by Western blot. Overnight cultures were diluted into fresh LB to give A₆₀₀=0.002, and growth was measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 17 and 24 h. Supernatant samples (6 ml) from each time point were filtered (0.22 μm Millex syringe-driven filter units. Millipore, Cork, Ireland) and concentrated 10-fold using Amicon Ultra-4 membranes (Millipore). Samples of 5 μl were mixed with 5 μl of 2× Tris-glycine SDS sample loading buffer (126 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.005% bromophenol blue) (Invitrogen), heated (96° C., 10 min) and separated on 4-12% Bis-Tris NuPAGE gels) using NuPAGE MOPS SDS running buffer (Invitrogen). Precision Plus Protein Standard (All Blue, Bio-Rad, Hercules, Calif.) was used as a molecular weight marker. Proteins were transferred to MagnaCharge 0.45 μm nylon membranes (Osmonics Inc., Minnetonka, Minn.) using transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol), blocked in PBS+3% skim milk (Difco, Lawrence, K S) for 1 h at room temperature, followed by three 10 min washes in PBST (PBS+0.05% Tween20). Membranes were then incubated with primary antibody diluted in PBS+1% skim milk overnight at 4° C. Mouse monoclonal antibody PA-05-A-G1 Lot#071100-02, 5.9 mg/ml (Naval Medical Research Center, Biological Defense Research Directorate), for detection of PA, and mouse monoclonal antibody LF-03-A-G1 Lot#150900-01, 7.4 mg/ml (NMRC, BDRD), for detection of LF, were both used at 1:2000. Rabbit antisera for development of blots included anti-EF serum #5900 (used at 1:8000; produced in inventors' laboratory), anti-camelysin serum (used at 1:2000; Molecular Biology Institute of Barcelona, Spain), and anti-recombinant ALO serum (used at 1:2000; R. Rest, Drexel University College of Medicine. Philadelphia, Pa.). Appropriate HRP-conjugated secondary IgGs (KPL, Gaithersburg, Md.) were used at 1:10000 followed by development with TMB (3,3′,5,5′-tetramethylbenzidine) (KPL).

Isolation and Purification of EF Protein

EF (B. anthracis edema factor) protein with an N-terminal six-histidine tag was expressed from plasmid pProEx-H6-EF (provided by Wei-Jen Tang) in E. coli BL21(DE3) (Promega) as previously described (Soelaiman S et al., 2003, J. Biol. Chem., 278, 25990-25997). pProEx-H6-EF comprises a nucleic acid molecule encoding EF with an N-terminal six-histidine tag operatively linked to pPROEX HTa (Addgene, Cambridge, Mass.).

EF was expressed in B. anthracis host strains from plasmid pSJ136EFOS (SEQ ID NO:62 and FIG. 6, which contains the EF structural gene in plasmid pYS5 under the control of (i.e., operatively linked to) the PA (B. anthracis protective antigen) promoter and signal sequence (Singh Y et al., 1989, J. Biol. Chem. 264, 19103-19107). Host strains BH450 and BH460 containing pSJ136EFOS were grown in FA medium containing 15 μg/ml of kanamycin at 37° C. for 14 h, essentially following procedures previously used for production of LF (Park S et al., 2000, Protein Expr. Purif. 18, 293-302). The cultures were cooled, supplemented with 2 μg/ml of AEBSF [4-(2-Aminoethyl)-benzenesulfonylfluoride.HCl] (US Biological, Swampscott, Mass.) and centrifuged at 4550×g for 30 min. All subsequent steps were performed at 4° C.

The supernatants were filter sterilized and supplemented with 5 mM EDTA. Solid ammonium sulfate was added to the supernatants to obtain 40% saturation. Phenyl-Sepharose Fast Flow (low substitution, GE Healthcare Biosciences Corp.) was added and supernatants gently mixed in the cold for 1.5 h. The resins were collected on porous plastic funnels (BelArt Plastics, Pequannock, N.J.) and washed with buffer containing 1.5 M ammonium sulfate, 10 mM Tris-HCl, and 1 mM EDTA (pH 8.0). The EF proteins were eluted with 0.3 M ammonium sulfate, 10 mM Tris-HCl, and 1 mM EDTA (pH 8.0), precipitated by adding an additional 30 g ammonium sulfate per 100 ml eluate, and centrifuged at 18,370×g for 20 min. The proteins were dissolved and dialyzed against 5 mM HEPES, 0.5 mM EDTA (pH 7.5). The dialyzed samples were applied to a Q-Sepharose Fast Flow column (GE Healthcare Biosciences Corp.) and eluted with a 0-0.5 M NaCl gradient in 20 mM Tris-HCl, 0.5 mM EDTA (pH 8.0). The EF-containing fractions identified by SDS-PhastGel analysis were purified on a column of ceramic hydroxyapatite (Bio-Rad Laboratories, Hercules, Calif.) with a gradient of 0.02-1.0 M potassium phosphate containing 0.1 M NaCl (pH 7.0). The fractions containing EF were dialyzed overnight against 5 mM HEPES and 0.5 mM EDTA, pH 7.5, concentrated as necessary, frozen, and stored at −80° C. The molecular mass of purified EF was estimated by liquid chromatogram-electrospray mass spectrometry using an HP/Agilent 1100 MSD instrument (Hewlett Packard, Palo Alto, Calif.) at the NIDDK core facility, Bethesda, Md.).

Analysis of EF Activity In Vitro and In Vivo

EF activity was measured by analysis of cyclic AMP (cAMP) production in the RAW264.7 macrophage cell line (ATCC TIB-71). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 2 mM Glutamax, 2 mM HEPES and 50 μg/ml gentamicin (all from Invitrogen) at 37° C. in 5% CO₂. Cells were seeded in 96-well plates 24 h prior to assays. EF preparations were serially diluted in a constant PA (B. anthracis protective antigen) concentration (250 ng/ml) prior to addition to cells and incubation for 1 h at 37° C. Total cAMP levels were assessed using the BioTRAK cAMP enzyme immunoassay kit (GE Healthcare Biosciences Corp.) according to the manufacturer's protocol. For analysis of EF potency, groups of five 8-week old female Balb/cJ mice (Jackson Laboratories, Bar Harbor, Me.) were injected via tail vein with EF preparations combined with an equal dose of PA. Toxin was prepared in sterile PBS. Mice were monitored for survival for 168 h. All experiments involving animals were performed under protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Example 2 Genetic Modification of B. anthracis Protease Genes

This Example demonstrates that a genetic modification of B. anthracis in which loxP is inserted into genes encoding B. anthracis proteases results in truncation of corresponding proteins.

A number of protease genes on the B. anthracis chromosome as well as the spo0A gene were targeted for inactivation in this study. They are identified here with the gene numbers assigned by The Institute for Genomic Research, now the J Craig Ventner Institute (Rockville, Md.), for the “Ames ancestor” strain chromosome (GenBank Accession No. NC 007530, gene designation GBAA_gene number) (Ravel J et al, 2009, J. Bacteriol. 191, 445-446). The gene numbers are coincident with the previous “Ames” strain chromosome (GenBank Accession No. NC 003997, gene designation BA_gene number) (Read T D et al., 2003, Nature 423, 81-86). All genes and proteins inactivated or analyzed in this study are listed in Table 1 together with corresponding locus tags.

The inactivation of the nprB and inhA1 genes has been previously described (Pomerantsev A P et al., 2006, ibid., Kastrup CJ, ibid.). The inhA2, tasA, calY, and mmpZ genes were inactivated as described herein. The B. anthracis InhA2 protein is 96% identical in sequence to the InhA2 protease of Bacillus thuringiensis, which is an essential virulence factor in that insect pathogen (Fedhila S et al., 2003, J. Bacteriol. 185, 2820-2825). The tasA gene (GBAA_1288) located downstream of the putative signal protease gene sipW (GBAA_1287) is only 5 genes upstream of the InhA1 gene (GBAA_1295). The intervening genes include the calY protease gene, and the two regulatory genes sinI and sinR (Table 4). The SinI and SinR proteins play important roles in B. subtilis biofilm formation, acting through protease-dependent processes (Chai Y, 2010, Mol. Microbiol. 78, 218-229), while in B. anthracis these proteins regulate secreted proteases (Pflughoeft K J et al., 2010, J. Bacteriol. 193, 631-639). It is interesting that the genes corresponding to calY and inhA1 are not found in the sinI, sinR region of the B. subtilis genome. Only the gene for the TasA protease, tasA, is located downstream of sipW and upstream of sinR and sinI (Pflughoeft et al., ibid.). Both TasA and camelysin are similar to the B. subtilis TasA protease (36% and 34% sequence identities, respectively). The final gene selected for inactivation, mmpZ, is reported to form an operon with the downstream gene (GBAA_3160) (Passalacqua K D et al., 2009, J. Bacteriol. 191, 3203-3211). The latter gene encodes a hypothetical secreted protein that is overproduced in B. anthracis (Antelmann H et al., ibid.). The absence of the MmpZ protease in the B. anthracis secretome indicates that this protease could be a target for proteolytic degradation by other proteases during the stationary phase of growth Antelmann H et al., ibid.).

The protease genes were inactivated as described in Example 1, followed by sequencing to locate the loxP insertions and infer the corresponding amino acid changes in the mutated proteins. These are shown in FIG. 1. Typically, the 34-bp loxP sequence will generate a frameshift and early downstream occurrence of a stop codon in an alternative reading frame either within the loxP sequence or soon thereafter. Thus, all four inactivated protease genes encoded greatly shortened proteins.

Example 3 Comparison of Protein Degradation by Genetically Modified B. anthracis and A35 Strain

This Example compares protein degradation by several genetically modified B. anthracis, including some B. anthracis of the embodiments, and the Ames 35 strain.

The levels of the three B. anthracis toxin components, ALO, and camelysin produced by the B. anthracis mutants were compared to those of the parental A35 strain by Western blot (FIG. 2). The ten strains were grown in LB at 37° C. over a 24-h period. All strains grew similarly except the six-protease mutant BH460, which appeared to have a slight lag before reaching exponential growth phase.

Expression of PA by Ames 35 was detectable starting at 5 h of growth. However, intact PA (83 kDa) disappeared by the 9th hour of growth due to proteolytic degradation. Inactivation of the InhA2 protease did not influence production of PA while inactivation of Spo0A or camelysin actually reduced the half-life of PA by 1-2 h. Inactivation of NprB, TasA, or MmpZ resulted in increased PA stability up to 10 h. However, PA produced by all these strains was completely degraded after 17 h. The most stable production of PA among the single knockout mutants was found in the InhA1 strain. PA was present even at 24 h of growth with this strain, although some degradation occurred. Surprisingly, the A35DM double mutant did not demonstrate enhanced PA production. PA degradation generally began at 6-7 h of growth and continued through 24 h. Strikingly, the A35HMS strain with six inactivated proteases produced PA with minimal to no degradation during the full 24 h of growth. Similar analyses were performed to follow production of the other B. anthracis toxin components, EF and LF. Intact EF (89 kDa) was found to be more vulnerable to degradation than PA, while LF (90 kDa) was quite stable when produced from most mutant strains. The levels of intact EF and LF, and the timing of their production, paralleled what was seen for PA for each mutant strain.

The enhanced breakdown of PA and EF found in the A35DM strain may be explained by increased production of camelysin in A35DM compared with A35 or the two corresponding single protease knockouts. Although both the NprB and InhA1 knockout strains had increased levels of camelysin, the double mutation produced what seems to be a greater than additive effect. The camelysin levels produced by the A35DM strain were similar to those by the Spo0A mutant strain, A35ΔSpo0A. Both strains produced camelysin (19 kDa) that remained stable over the 24-h period. These observations on post-translational regulation of camelysin production by several proteases support and expand recently published data demonstrating that the concentration of InhA1 in culture supernatants is inversely proportional to the concentration of camelysin (Pflughoeft et al., ibid.). Another interesting finding of these studies was that the global transcriptional regulator Spo0A inhibited camelysin production. Pflughoeft et al. ibid., recently demonstrated that B. anthracis sinR (which was deleted in those mutants containing the tasA-ihhA1 deletion) also negatively regulates transcription of camelysin and InhA1, both of which have been suggested to be associated with virulence (Chung M C et al., ibid., Liu Y T et al., 2008, Protein Expr. Purif. 57, 72-80).

Knocking out TasA also increased production of camelysin but to a lesser extent than elimination of NprB and InhA1. The InhA2 knockout did not result in any change in camelysin degradation or production when compared to A35, while MmpZ elimination was actually detrimental to camelysin production. These studies indicate that inactivation of six proteases in B. anthracis leads to increased production of intact toxin proteins in culture supernatants relative to A35, single, or double protease mutants.

Very low levels of ALO were produced from the A35 strain compared to all the protease knockouts, with the exception of the InhA2 mutant. The ALO produced by the A35 strain completely disappeared after 9 hours of growth. Every protease knockout strain showed increased levels of production or greater stability of ALO (53-kDa band). It is interesting that ALO breakdown started in A35DM (less in A35ΔInhA1) only after 17 h of growth. A similar effect was not seen in the A35HMS strain, which allowed accumulation of intact ALO throughout the 24 h of growth. The ALO gene is under control of a PlcR-dependent promoter, so that the truncation of the PlcR protein in B. anthracis is expected to greatly limit ALO synthesis, along with all the other PlcR-dependent proteins (Sastalla, 2010, Microbiology 156, 2892-2993). The fact that ALO can be observed in several of the protease-deficient mutants implies that previous reports of low ALO production may be attributed, at least in part, to its degradation rather than to low expression. Camelysin overexpression did not influence ALO production, indicating that this toxin is not a target for camelysin. Taken together, the results demonstrate the involvement of multiple proteases in controlling the accumulation of extracellular proteins in B. anthracis.

Example 4 Complementation of Genetic Modifications to Inactivate a B. anthracis Protease

This Example demonstrates that complementation of genetic modifications to inactivate a B. anthracis protease restores proteolytic activity.

To verify that the changes observed above were due to the intended gene knockouts rather than to unrecognized second-site mutations, studies to assess complementation of several mutants were conducted. To complement the mmpZ mutation, the A35ΔMmpZ strain was transformed with pMmpZC. This plasmid undergoes a single crossover to insert the full length, wild-type mmpZ gene next to the mutated one. The pSC vector was eliminated by Cre-recombinase treatment as described previously (Pomerantsev A P et al., 2009, ibid.). The presence of the intact mmpZ gene in A35MmpZC was confirmed by PCR and sequencing. Western blot analysis of LF production from A35ΔMmpZC (FIG. 3A) over 24 h indicated that proteolytic activity was restored to levels similar to that of A35 (FIG. 2). To restore the large tasA-inhA1 deletion in the genome of the A35TM strain, the strain was transformed with the plasmid pΔTasA-InhA1C (containing the entire tasA-inhA1 region) and complementation was assessed in a manner similar to that described above. Analysis of ALO production from the complemented strain verified restoration of proteolytic activity (FIG. 3B). Complementation of the A35ΔInhA2 strain, and restoration of the InhA2 protease in the same manner, however, did not result in any difference in secreted proteins (data not shown).

All the studies described above were done in derivatives of Ames 35, where the toxin proteins are encoded on the large virulence plasmid pXO1. Production of the toxin proteins and secreted proteases in these strains is highly dependent on the growth medium. In particular, the production of the three toxin proteins and certain proteases is greatly enhanced by the addition of bicarbonate (Chitlaru T et al., ibid., Passalacqua K D et al., ibid., Bartkus J M, 1989, Infect. Immun. 57, 2295-2300. Thus, it is likely that growth in certain media could produce higher concentrations of both proteases and substrates (e.g., PA. LF, EF, etc.), and this could lead to even greater degradation than observed here.

Bacillus host strains are widely used in biotechnological processes, and avirulent B. anthracis strains have been used for a number of years to produce PA and LF (see, for example, Varughese M et al., ibid., Park S et al., ibid., Gupta P K et al., 2008, PLoS. ONE 3, e3130. However, these strains have not been useful as generic hosts for recombinant protein production due to the secreted proteases demonstrated by the work presented above. The successful elimination of many of the most abundant proteases described herein suggested that the resulting strains could have value as protein expression hosts. To create an optimal host, the A351-HMS strain was further modified by curing it of plasmid pXO1. The resulting strain, designated BH460, is non-toxigenic, and can be considered innocuous since it lacks the major virulence factors of B. anthracis. The permanent deletion of the spo0A gene assures that the strain dies rapidly at the end of exponential growth, eliminating concerns regarding laboratory contamination.

Example 5 Production of Intact EF Protein by B. anthracis BH460

This Example demonstrates production of intact EF by a modified B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSJ136EFOS.

Production of EF from B. anthracis hosts has previously been difficult because this protein is more susceptible to proteolytic degradation than are PA and LF. The plasmid pSJ136EFOS (the nucleic acid sequence of which is SEQ ID NO:62) encodes the mature EF protein with its native N-terminus (thus, the “OS” for original sequence) fused to the PA signal sequence and under the control of the pag promoter. This plasmid is otherwise similar to the plasmids pYS5 and pSJ115 that are routinely used by the inventors to produce PA and LF, respectively; see, e.g., Park S et al., ibid. Protein purified from the transformant BH460 (pSJ136EFOS) was compared to a preparation made in a similar way from the single protease mutant host BH450, and to a His6-tagged EF protein purified from E. coli (Soelaiman S et al., ibid.), the latter being the type of material used in previous toxicity analyses reported in Firoved A M et al., 2005, Am. J. Pathol. 167, 1306-1320. SDS-PAGE profiles of the recombinant EF proteins are shown in FIG. 4A. The EF produced from BH460 appeared to be slightly less degraded than that isolated from BH450. This finding was confirmed by mass-spectrometry analyses (FIG. 4B). The molecular mass of the recombinant protein isolated from BH460 (88,820 Da) compared well with the theoretical molecular weight for EF (88,822 Da), differing by 2 Da, which is within the instrumental error. A second species was found that had a lower mass (88,687 Da) consistent with loss of the N-terminal methionine. These two protein species were present in about equal amounts (47% for the larger, 53% for the smaller). Mass spectra of the EF produced from BH450 showed degradation as indicated by losses of 1495.7 and 2455.7 Da, resulting in proteins of 87,326 and 86,366 Da, found with similar abundances of 54% and 46%, respectively (FIG. 4C). Apparent protease cleavage sites in mature EF produced by BH450 were mapped to amino acid residues Arg-12 and Lys-20. EF purified from E. coli was monomorphic, with a mass of 89,995 Da, differing by only 6 Da from the theoretical molecular weight (89,989 Da, FIG. 4C). These results clearly demonstrate production of intact EF from BH460 compared to the truncated proteins made by BH450.

Example 6 Activity of EF Produced by Genetically Modified Protease-Deficient B. anthracis

This Example demonstrates the EF produced by a modified B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSJ136EFOS, is active in a potency assay.

As noted above, recombinant EF has previously been difficult to produce from B. anthracis host strains transduced with plasmids such as pSJ136. Furthermore, the EF that was obtained either from B. anthracis Sterne strain culture supernatant (Leppla S H, 1991, Methods Enzymol. 195, 153-168) or E. coli (Soelaiman S et al., ibid.) consistently had higher potency in inducing cAMP production in cultured cells or lethality to mice than EF produced from B. anthracis (data not shown). In fact, no previous recombinant EF preparations from B. anthracis have been lethal to mice even when injected in doses as high as 100 μg (combined with equimolar PA) (data not shown). The ES-MS analyses shown in FIG. 4 suggest that the low potency of previous B. anthracis-derived EF preparations could be due to degradation. Consistent with prior results, the BH450-derived EF displayed an extremely low level of specific activity and was not lethal for mice (FIG. 5A and FIG. 5B). However, the recombinant EF purified from the BH460 culture supernatant had a specific activity exceeding that of highly active E. coli BL21(DE3)-derived EF (FIG. 5A). Similarly, when injected with equal doses of PA, recombinant EF prepared from BH460 was lethal to animals at the 25 μg dose, whereas this dose of E. coli-derived EF had minimal effect (FIG. 5B). EF purified from BH450 was not lethal at 50 μg (FIG. 5B) and even at doses up to 100 μg (data not shown). Thus, the BH460 strain, which produces 5-7 mg of EF per liter of culture, allows for the first time the purification of substantial amounts of highly active EF from B. anthracis. Because other proteins purified in the same way from B. anthracis have consistently been free of endotoxin, use of these EF preparations also eliminates concerns regarding endotoxin-mediated cAMP co-signaling associated with E. coli-derived preparations. The BH460 strain has also proven very useful for expression of a variety of other proteins, typically yielding in excess of 10 mg of final pure protein per liter of culture, an Example of which is provided in Example 7.

Example 7 Production of Intact B. cereus Hemolysin HBL Toxins by B. anthracis BH460

This Example demonstrates that modified a B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSW4-HBL L1 His, pSW4-HBL L2 His, or pSW4-HBL B His, produces large quantities of intact HBL L1, HBL L2, or HBL B, respectively.

B. cereus can cause infections in immune-compromised patients, such as sepsis, meningitis, pneumonia, and wound infections. There are also reports on impetigo-like lesions in non-compromised patients. HBL toxins have been shown to contribute to B. cereus caused diarrheal food poisoning, and their presence is used as an indicator for B. cereus food contaminations.

B. cereus 569 has two HBL operons. The first operon is located on the chromosome, and the second on a plasmid. The chromosomal operon appears to comprise three genes that encode the following proteins: L2 (1320 bp, 439 amino acids, 49.3 kDa with signal sequence); L1 (1221 bp, 406 amino acids, 43.8 kDa with signal sequence); and B (1146 bp, 381 amino acids, 42.5 kDa with signal sequence). These proteins do not share high homology on the protein level: L1 vs. L2 has 24% identity; L1 vs B has 26% identity; and L2 vs B also has 26% identity. All of these HBL proteins have been shown to be secreted via the Sec pathway and contain a Gram-positive signal sequence (Fagerlund A et al., 2010, BMC Microbiol 10, 304).

Single HBL toxin components are non-toxic; all three components (i.e., L1, L2, and B) are needed to effect cell lysis. The exact mechanism by which the toxin components function in concert is still not well understood.

HBL has been shown to affect a variety of cell types and tissues such as retinal tissue (Beecher D J et al., 1995, Infect. Immun. 63, 4423-4428), rabbit skin, ileum, CHO cells (Beecher D J et al., 1997, J. Biol. Chem. 272, 233-239), and red blood cells from guinea pig, swine, bovine, sheep, rabbit, goat, and human (activity in descending order). Studies performed by Beecher et al., 1997, ibid. showed that the B (binding) moiety can prime erythrocytes to lyse when followed by incubation with L1 and L2 components. Cells can also be primed with either L component, indicating that all three components can bind to erythrocyte membranes. In addition, toxin action can be inhibited by addition of antibodies specific to the binding component, as well as by addition of excess L1 component, indicating that L1 binds either L2 or the binding component (Beecher et al., 1997, ibid.).

Recombinant molecule pSW4-HBL L1 His (depicted in FIG. 7) was produced by operatively linking a nucleic acid molecule encoding HBL L1 (including its natural signal sequence) and a C-terminal 6×His tag to expression vector pSW4, such that expression of HBL L1 His was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL L1 His, and the modified B. anthracis, when grown in 2 liters FA broth overnight (˜16 h), produced 28 mg of pure HBL L1 His protein.

Recombinant molecule pSW4-HBL L2 His (depicted in FIG. 8) was produced by operatively linking a nucleic acid molecule encoding HBL L2 (including its natural signal sequence) and a C-terminal 6×His tag to expression vector pSW4, such that expression of HBL L2 His was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL L2 His, and the modified B. anthracis, when grown in 2 liters FA broth overnight (˜16 h), produced 151 mg of pure HBL L2 His protein.

Recombinant molecule pSW4-HBL B His (depicted in FIG. 9) was produced by operatively linking a nucleic acid molecule encoding HBL B (including its natural signal sequence) and a C-terminal 6×His tag to expression vector pSW4, such that expression of HBL B H is was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL B His, and the modified B. anthracis, when grown in 2 liters of FA broth overnight (−16 h), produced 75 mg of pure HBL B His protein.

Purification of the respective proteins was accomplished by the following steps: adsorbed protein to phenyl-Sepharose at 2 M ammonium sulfate; eluted protein from phenyl-Sepharose with 0.3 M ammonium sulfate; precipitated protein, resuspended, and dialyzed against 5 mM HEPES, 0.5 mM EDTA, pH 7.5 prior to loading onto a Q-Sepharose column (GE Healthcare); eluted protein from Q-Sepharose using a NaCl gradient (buffer A was 20 mM Tris-HCl, 0.5 mM EDTA, pH 8.0; buffer B was 20 mM Tris-HCl, 0.5 mM EDTA, pH 8.0, plus 0.5 M NaCl); combined protein-containing fractions and dialyzed against 5 mM HEPES, 0.5 mM EDTA, pH 7.5 prior to loading onto a hydroxyapatite (BioRad ceramic type) column; eluted protein from hydroxyapatite using a phosphate gradient (buffer A was 0.02 mM potassium phosphate, pH 7.0, 0.10 M NaCl; buffer B was 1 M potassium phosphate, pH 7.0, 0.10 M NaCl); collected protein peak; and dialyzed pure protein prior to concentration on an ultrafiltration unit.

Example 8 Genetic Modification of B. anthracis Protease Genes

This Example demonstrates that genetic modification of a gene encoding a protease protein in B. anthracis using a Flp-FRT recombinase system results in truncation of the corresponding protein and production of a B. anthracis strain that no longer expresses such active protease.

Culture supernatants of strain BH460 were analyzed by mass spec to determine which proteases continued to be produced that might degrade valuable endogenous proteins (e.g., anthrax toxin proteins that are vaccine candidates) or heterologous proteins. Two additional proteases were identified (GBAA_1995 cysteine protease CysP1 and GBAA_4584 minor extracellular protease VpR). At least a portion of genes encoding these proteases were genetically deleted from B. anthracis BH460, leading to B. anthracis BH480, which lacks eight active proteases, due to genetic modification of the following genes leading to inactivation of the respectively encoded proteins: nprB, inhA2, tasA, calY, inhA1, mmpZ, cysP1, and vpR. Mutations engineered into B. anthracis cysP1 and vpR are indicated in FIG. 1C.

Specifically, a Saccharomyces cerevisiae Flp-FRT recombinase system (Park, Y N, et al., 2011, Yeast 28, 673-681) was adapted to use for B. anthracis cysP1 and vpR gene deletions by introducing S. cerevisiae Flp recombinase and S. cerevisiae FRT sites into the genome of B. anthracis; both FRT sites and Flp-recombinase genes were inserted into vectors suitable for replication, expression, and recombination in B. anthracis. The system was used in a manner similar to the Cre-loxP recombinase system described previously (Pomerantsev A P et al., 2009, ibid.) and herein. The Flp-FRT method allowed the replacement of both cysP1 and vpR genes with a 48-bp FRT-site using Flp recombinase. Both FRT-site and flp-genes were cloned into plasmids pSCF and pFPAS, respectively, which are described in Table 3. Additional plasmids used in the genetic modifications of the cysP1 and vpR genes included plasmid pCysP1L1, pCysP1RI, pVpRLI, and pVpRR1, which are described in Table 3. Primer pair 1995 seqF and 1995 seqR was used to verify cysP1 gene disruption; and primer pair 4584 seqF and 4584 seqR was used to verify vpR gene disruption. Table 5 provides mass spec (MS) analysis that confirms the absence of both CysP1 and VpR proteases in the secretome of the B. anthracis BH480 strain.

TABLE 5 MS-peptide hits for CysP1 and VpR proteases in supernatants of B. anthracis BH460 and B. anthracis BH480. Protease BH460 BH480 GBAA_1995_CysP1 69 0 GBAA_4584_VpR 36 0

Table 10 demonstrates the absence of both CysP1 and VpR proteases in the supernatant of B. anthracis BH480 in comparison to that of B. anthracis BH460.

Supernatants of BH480 have been examined as described above, and several additional proteases have been identified: (GBAA_2183 neutral metalloprotease NprC, GBAA_5414 serine protease SprA, GBAA_3660 serine protease HtrA and GBAA_3968 prokaryotic homolog of proteasome HslV).

Example 9 Production of Intact EF, LF, and uPA Proteins by B. anthracis BH480

This Example demonstrates production of EF proteins by modified B. anthracis of the embodiments, namely B. anthracis BH480 transformed with recombinant molecules encoding a variety of EF proteins. Also demonstrated is production of intact LF and uPA proteins.

B. anthracis BH480 was transformed with recombinant molecule pSJ136EFOS in a manner similar to transformation of B. anthracis BH460 with recombinant molecule pSJ136EFOS as described herein. B. anthracis BH480 was also transformed with recombinant molecule pSJ136EF-His, recombinant pSJ136EF-Cys, or recombinant molecule pSJ136EF-NEHY, which are identical to pSJ136EFOS except that they encode mature EF proteins having small modifications in the N-terminal sequence as indicated in Table 6.

TABLE 6 Recombinant molecules and proteins encoded by such recombinant molecules Recombinant Encoded EF N-terminus of molecule protein EF protein pSJ136EFOS EFOS (SEQ ID MNEHY . . . NO: 64) pSJ136EF-His EF-His (SEQ ID HNEHY . . . NO: 93) pSJ136EF-Cys EF-Cys (SEQ ID CNEHY . . . NO: 94) pSJ136EF-NEHY EF-NEHY (SEQ ID NEHY . . . NO: 95)

B. anthracis strains transformed with pSJ136EFOS, pSJ136EF-His, pSJ136EF-Cys, or pSJ136EF-NEHY were inoculated into 10 ml FA media that included 10 micrograms per ml (μg/ml) kanamycin. Protein production for each sample was demonstrated by native Phast gel (native 8-25% acrylamide gradient) analysis, the proteins being stained by Coomassie blue. Results are shown in FIG. 10. The results indicate production of intact EF proteins by B. anthracis BH480 transformed with pSJ136EFOS, pSJ136EF-His, pSJ136EF-Cys, or pSJ136EF-NEHY, as shown in lanes 4, 6, 7, and 8 of FIG. 10, respectively.

FIG. 10 also shows production of intact urokinase plasminogen activator (uPA) variants PA-U2f (lane 1) and PA-U7f (lane 2) by B. anthracis BH480 transformed with pYS5 plasmids encoding PA-U2f and PA-U7f, respectively, and cultured as described herein; PA-U2, PA-U7, and recombinant molecules comprising nucleic acid molecules encoding such proteins are described in International Publication No. WO 01/21656, published 29 Mar. 2001; PA-U2f and PA-U7f lack a C-terminal serine present in PA-U2 and PA-U7.

FIG. 10, lane 3 shows production of intact mature lethal factor (LF-OS) by B. anthracis BH480 transformed with pSJ115-LF-OS and cultured as described herein. Lane 5 shows that B. anthracis BH480 transformed with pSJ136 Hfq1-FLAG and cultured as described herein did not produce detectable amounts of protein. Hfq1-FLAG is a small RNA chaperone Hfq1 with a C-terminal FLAG tag.

Example 10 Production of Intact Recombinant Proteins by B. anthracis BH480

This Example demonstrates production of recombinant proteins by modified B. anthracis of the embodiments.

Intact fusion protein LFnBlaY was produced as follows: B. anthracis BH480 was transformed with a recombinant molecule comprising a pSJ115 plasmid encoding LFnBlaY. LFnBlaY is a fusion protein of B. anthracis lethal factor LFn to B. cereus beta-lactamase (BlaY) that has an amino acid sequence represented by SEQ ID NO:91. A similar fusion protein, LF-BLA, produced in E. coli, is described in Hobson J P et al., 2006, Nature Methods 3, 259-261. The modified B. anthracis was cultured and LFnBlaY purified as described herein. A 2-liter preparation yielded 184 mg of intact LFnBlaY, the purity of which is demonstrated in FIG. 11, lanes 4 and 5, and compared to the purity of LF-BLA from E. coli (lane 3).

Intact protective antigen (PA) variant PA-SNKE-deltaFF-E308D was produced as follows: B. anthracis BH480 was transformed with a recombinant molecule comprising a pYS5 plasmid encoding PA-SNKE-deltaFF-E308D. PA-SNKE-deltaFF-E308D is described in Ramirez D M et al., 2002, J Industrial Microbiology & Biotechnology 28, 232-238. The PA variant's amino acid sequence is represented by amino acid sequence SEQ ID NO:92. The modified B. anthracis was cultured and PA-SNKE-deltaFF-E308D purified using techniques similar to those described in US 2004/0076638 A1, published Apr. 22, 2004. A 2-liter preparation yielded 122 mg of intact PA-SNKE-deltaFF-E308D, the purity of which is shown in FIG. 5, lane 2.

This Example along with other examples herein demonstrate the abilities of B. anthracis BH460 and B. anthracis BH480 to make a variety of intact recombinant proteins.

Example 11 Summary and Conclusions

These Examples describe the adaptation of an improved Cre-loxP system for sequentially deleting additional protease-encoding genes of B. anthracis. They also describe a role of each protease in degradation of B. anthracis toxin components and another potential virulence factor, anthrolysin O (ALO) (Shannon J G et al., 2003, Infect. Immun. 71, 3183-318).

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. This study shows that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (NprB, InhA2, TasA, camelysin, InhA1, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or E. coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture.

These Examples also describe the successful adaptation of a modified Saccharomyces cerevisiae Flp-FRT recombinase system for deleting additional protease-encoding genes of B. anthracis. This system was used to inactivate CysP1 and VpR proteases in BH460, thereby creating B. anthracis BH480. BH480 is also recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure intact protein per liter of culture. In some embodiments, the yield is greater than 50 mg pure intact protein per liter of culture. In some embodiments, the yield is greater than 90 mg pure intact protein per liter of culture.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims. 

What is claimed is:
 1. A Bacillus anthracis (B. anthracis) transformed with a recombinant molecule encoding a product, wherein: (a) the B. anthracis comprises genetic modifications that inactivates proteases NprB encoded by a genetically modified nprB gene at locus GBAA_0599 of B. anthracis, InhA2 encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis, TasA encoded by a genetically modified tasA4 gene at locus GBAA_1288 of B. anthracis, Camelysin encoded by a genetically modified calY gene at locus GBAA_1290 of B. anthracis, InhA1 encoded by a genetically modified inhA1 gene at locus GBAA_1295 of B. anthracis and MmpZ encoded by a genetically modified mmpZ gene at locus GBAA_3159 of B. anthracis, and the B. anthracis is selected from the group consisting of a B. anthracis that is sporulation-deficient, a B. anthracis that lacks virulence plasmid pXO1, virulence plasmid pXO2, or virulence plasmids pXO1 and pXO2, and a B. anthracis that is sporulation-deficient and lacks virulence plasmid pXO1, virulence plasmid pXO2, or virulence plasmids pXO1 and pXO2; and (b) the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector, wherein the product comprises an intact protein.
 2. The B. anthracis of claim 1, wherein the product is a toxin.
 3. The B. anthracis of claim 2, wherein the toxin is selected from the group consisting of an anthrax toxin, a cholera toxin, a diphtheria toxin, a hemolysin, a ricin, a Pseudomonas toxin, a Haemophilus ducreyi toxin, an Escherichia coli toxin, and a ribosome-inactivating protein (RIP) toxin.
 4. The B. anthracis of claim 1, wherein the intact protein is selected from the group consisting of an anthrax edema factor (EF), an anthrax lethal factor (LF), an anthrax protective antigen (PA), anthrolysin (ALO), a Bacillus cereus hemolysin, and combinations thereof.
 5. A method to produce a product comprising: (a) culturing the B. anthracis of claim 1 in a medium to produce the product; and (b) recovering the product.
 6. The B. anthracis of claim 1, wherein the B. anthracis is derived from strain Ames 35 (pXO1⁺pXO2⁻).
 7. The B. anthracis of claim 1, wherein the B. anthracis is sporulation-deficient and lacks virulence plasmids pXO1 and pXO2.
 8. The B. anthracis of claim 1, wherein the B. anthracis is BH460. 